Processes for preparation of 9,11-epoxy steroids and intermediates useful therein

ABSTRACT

Multiple reaction schemes, process steps and intermediates are provided for the synthesis of epoxymexrenone, useful as a diuretic, and other 9,11-epoxy-steroids.

This application claims benefit of U.S. Provisional Application No.60/008,455, filed Dec. 11, 1995.

BACKGROUND OF THE INVENTION

This invention relates to the novel processes for the preparation of9,11-epoxy steroid compounds, especially those of the 20-spiroxaneseries and their analogs, novel intermediates useful in the preparationof steroid compounds, and processes for the preparation of such novelintermediates. Most particularly, the invention is directed to novel andadvantageous methods for the preparation of methyl hydrogen9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone(eplerenone; epoxymexrenone).

Methods for the preparation of 20-spiroxane series compounds aredescribed in U.S. Pat. No. 4,559,332. The compounds produced inaccordance with the process of the '332 patent have an open oxygencontaining ring E of the general formula: ##STR1## in which --A--A--represents the group --CH₂ --CH₂ -- or --CH═CH--,

R¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonylradical.

--B--B-- represents the group --CH₂ --CH₂ -- or an α- or β-orientedgroup ##STR2## R⁶ and R⁷ being hydrogen X represents two hydrogen atomsor oxo,

Y¹ and Y² together represent the oxygen bridge --O--, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy,

and salts of such compounds in which X represents oxo and Y² representshydroxy, that is to say of corresponding 17β-hydroxy-21-carboxylicacids.

U.S. Pat. No. 4,559,332 describes a number of methods for thepreparation of epoxymexrenone and related compounds of Formula IA. Theadvent of new and expanded clinical uses for epoxymexrenone create aneed for improved processes for the manufacture of this and otherrelated steroids.

SUMMARY OF THE INVENTION

The primary object of the present invention is the provision of improvedprocesses for the preparation of epoxymexrenone, other 20-spiroxanes andother steroids having common structural features. Among the particularobjects of the invention are: to provide an improved process thatproduces products of Formula IA and other related compounds in highyield; the provision of such a process which involves a minimum ofisolation steps; and the provision of such a process which may beimplemented with reasonable capital expense and operated at reasonableconversion cost.

Accordingly, the present invention is directed to a series of synthesisschemes for epoxymexrenone; intermediates useful in the manufacture ofeplerenone; and syntheses for such novel intermediates.

The novel synthesis schemes are described in detail in the Descriptionof Preferred Embodiments. Among the novel intermediates of thisinvention are those described immediately below.

A compound of Formula IV corresponds to the structure: ##STR3## wherein:--A--A-- represents the group --CHR⁴ --CHR⁵ -- or CR⁴ ═CR⁵ --

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halos hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxy carbonyl, cyano, aryloxy,

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical,

R² is an 11α-leaving group the abstraction of which is effective forgenerating a double bond between the 9- and 11-carbon atoms;

--B--B-- represents the group --CHR⁶ --CHR⁷ -- or an alpha- orbeta-oriented group: ##STR4## where R⁶ and R⁷ are independently selectedfrom the group consisting of hydrogen, halo, lower alkoxy, acyl,hydroxalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

A compound of Formula IVA corresponds to Formula IV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR5## where x, Y¹, Y² and C(17) are as defined above.

A compound of Formula IVB corresponds to Formula IVA wherein R⁸ and R⁹together form the structure of Formula XXXIII: ##STR6##

Compounds of Formulae IVC, IVD and IVE, respectively, correspond to anyof Formula IV, IVA, or IVB wherein each of --A--A-- and --B--B-- is--CH₂ --CH₂ --, R³ is hydrogen, and R¹ is alkoxycarbonyl, preferablymethoxycarbonyl. Compounds within the scope of Formula IV may beprepared by reacting a lower alkylsulfonylating or acylating reagent, ora halide generating agent, with a corresponding compound within thescope of Formula V.

A compound of Formula V corresponds to the structure: ##STR7## wherein--A--A--, --B--B--, R¹, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VA corresponds to Formula V wherein R⁸ and R⁹ withthe ring carbon to which they are attached together form the structure:##STR8## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VB corresponds to Formula VA wherein R⁸ and R⁹together form the structure of Formula XXXIII: ##STR9##

Compounds of Formulae VC, VD and VE, respectively, correspond to any ofFormula V, VA, or VB wherein each of --A--A-- and --B--B-- is --CH₂--CH₂ --, R³ is hydrogen, and R¹ is alkoxycarbonyl, preferablymethoxycarbonyl. Compounds within the scope of Formula V may be preparedby reacting an alkali metal alkoxide with a corresponding compound ofFormula VI.

A compound of Formula VI corresponds to the structure: ##STR10## wherein--A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIA corresponds to Formula VI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR11## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIB corresponds to Formula VIA wherein R⁸ and R⁹together form the structure of Formula XXXIII: ##STR12##

Compounds of Formulae VIC, VID and VIE, respectively, correspond to anyof Formula VI, VIA, or VIB wherein each of --A--A-- and --B--B-- is--CH₂ --CH₂ --, and R³ is hydrogen. Compounds of Formula VI, VIA, VIBand VIC are prepared by hydrolyzing a compound corresponding to FormulaVII, VIIA, VIIB or VIIC, respectively.

A compound of Formula VII corresponds to the structure: ##STR13##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIA corresponds to Formula VII wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR14## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIB corresponds to Formula VIIA wherein R⁸ and R⁹together form the structure of Formula XXXIII: ##STR15##

Compounds of Formulae VIIC, VIID and VIIE, respectively, correspond toany of Formula VII, VIIA, or

VIIB wherein each of --A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ ishydrogen. A compound within the scope of Formula VII may be prepared bycyanidation of a compound within the scope of Formula VIII.

A compound of Formula VIII corresponds to the structure: ##STR16##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIIA corresponds to Formula VIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure: ##STR17## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIIB corresponds to Formula VIIIA wherein R⁸ andR⁹ together form the structure of Formula XXXIII: ##STR18##

Compounds of Formulae VIIIC, VIIID and VIIIE, respectively, correspondto any of Formula VIII, VIIIA, or VIIIB wherein each of --A--A-- and--B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compounds within thescope of Formula VIII are prepared by oxidizing a substrate comprising acompound of Formula XXX as described hereinbelow by fermentationeffective for introducing an 11-hydroxy group into the substrate inα-orientation.

A compound of Formula XIV corresponds to the structure: ##STR19##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XIVA corresponds to Formula XIV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR20## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XIV corresponds to Formula XIVA wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII: ##STR21##

Compounds of Formulae XIVC, XIVD and XIVE, respectively, correspond toany of Formula XIV, XIVA, or XIVB wherein each of --A--A-- and --B--B--is --CH₂ --CH₂ --, and R³ is hydrogen. Compounds within the scope ofFormula XIV can be prepared by hydrolysis of a corresponding compoundwithin the scope of Formula XV.

A compound of Formula XV corresponds to the structure: ##STR22## wherein--A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XVA corresponds to Formula XV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR23## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XVB corresponds to Formula XVA wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII: ##STR24##

Compounds of Formulae XVC, XVD and XVE, respectively, correspond to anyof Formula XV, XVA, or XVB wherein each of --A--A-- and --B--B-- is--CH₂ --CH₂ --, and R³ is hydrogen. Compounds within the scope ofFormula XV can be prepared by cyanidation of a corresponding compoundwithin the scope of Formula XVI.

A compound of Formula XXI corresponds to the structure: ##STR25##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIA corresponds to Formula XXI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure: ##STR26## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIB corresponds to Formula XXIA wherein R⁸ and R⁹together form the structure of Formula XXXIII: ##STR27##

Compounds of Formulae XXIC, XXID and XXIE, respectively, correspond toany of Formula XXI, XXIA, or XXIB wherein each of --A--A-- and --B--B--is --CH₂ --CH₂ --, and R³ is hydrogen Compounds within the scope ofFormula XXI may be prepared by hydrolyzing a corresponding compoundwithin the scope of Formula XXII.

A compound of Formula XXII corresponds to the structure: ##STR28##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIA corresponds to Formula XXII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure: ##STR29## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIB corresponds to Formula XXIIA wherein R⁸ andR⁹ together form the structure of Formula XXXIII: ##STR30##

Compounds of Formulae XXIIC, XXIID and XXIIE, respectively, correspondto any of Formula XXII, XXIIA, or XXIIB wherein each of --A--A-- and--B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compounds within thescope of Formula XXII may be prepared by cyanidation of a compoundwithin the scope of Formula XXIII.

A compound of Formula XXIII corresponds to the structure: ##STR31##wherein --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIIA corresponds to Formula XXIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure: ##STR32## where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIIB corresponds to Formula XXIIIA wherein R⁸and R⁹ together form the structure of Formula XXXIII: ##STR33##

Compounds of Formulae XXIIIC, XXIIID and XXIIIE, respectively,correspond to any of Formula XXIII, XXIIIA, or XXIIIB wherein each of--A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compoundswithin the scope of Formula XXIII can be prepared by oxidation of acompound of Formula XXIV, as described hereinbelow.

A compound of Formula 104 corresponds to the structure: ##STR34##wherein --A--A--, --B--B--, and R³ are as defined in Formula IV, and R¹¹is C₁ to C₄ alkyl.

A compound of Formula 104A corresponds to Formula 104 wherein each of--A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compoundswithin the scope of Formula 104 may be prepared by thermal decompositionof a compound of Formula 103.

A compound of Formula 103 corresponds to the structure: ##STR35##wherein --A--A--, --B--B--, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 103A corresponds to Formula 103 wherein each of--A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compoundswithin the scope of Formula 103 may be prepared by reaction of acorresponding compound of Formula 102 with a dialkyl malonate in thepresence of a base such as an alkali metal alkoxide.

A compound of Formula 102 corresponds to the structure: ##STR36##wherein --A--A--, --B--B--, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 102A corresponds to Formula 102 wherein each of--A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compoundswithin the scope of Formula 102 may be prepared by reaction of acorresponding compound of Formula 101 with a trialkyl sulfonium compoundin the presence of a base.

A compound of Formula 101 corresponds to the structure: ##STR37##wherein --A--A--, --B--B--, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 101A corresponds to Formula 101 wherein each of--A--A-- and --B--B-- is --CH₂ --CH₂ --, and R³ is hydrogen. Compoundswithin the scope of Formula 101 may be prepared by reaction of11α-hydroxyandrostene-3,17-dione or other compound of Formula XXXVI witha trialkyl orthoformate in the presence of an acid.

Based on the disclosure of specific reaction schemes as set outhereinbelow, it will be apparent which of these compounds have thegreatest utility relative to a particular reaction scheme. Use of thecompounds of this invention are useful as intermediates forepoxymexrenone and other steroids.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow sheet of a process for the bioconversion ofcanrenone or a canrenone derivative to the corresponding 11α-hydroxycompound;

FIG. 2 is a schematic flow sheet of a preferred process for thebioconversion of 11-α-hydroxylation of canrenone and canrenonederivatives;

FIG. 3 is a schematic flow sheet of a particularly preferred process forthe bioconversion of 11-α-hydroxylation of canrenone and canrenonederivatives;

FIG. 4 shows the particle size distribution for canrenone as prepared inaccordance with the process of FIG. 2; and

FIG. 5 shows the particle size distribution for canrenone as sterilizedin the transformation fermenter in accordance with the process of FIG.3.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, various novel process schemeshave been devised for the preparation of epoxymexrenone and othercompounds corresponding Formula I: ##STR38## wherein: --A--A--represents the group --CHR⁴ --CHR⁵ -- or --CR⁴ ═CR⁵ --

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkylradical,

--B--B-- represents the group --CHR⁶ --CHR⁷ -- or an alpha- orbeta-oriented group: ##STR39## where R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen, halo, lower alkoxy,acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

Unless stated otherwise, organic radicals referred to as "lower" in thepresent disclosure contain at most 7, and preferably from 1 to 4, carbonatoms.

A lower alkoxycarbonyl radical is preferably one derived from an alkylradical having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec.-butyl and tert.-butyl; especiallypreferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl. Alower alkoxy radical is preferably one derived from one of theabove-mentioned C₁ -C₄ alkyl radicals, especially from a primary C₁ -C₄alkyl radical; especially preferred is methoxy. A lower alkanoyl radicalis preferably one derived from a straight-chain alkyl having from 1 to 7carbon atoms; especially preferred are formyl and acetyl.

A methylene bridge in the 15,16-position is preferably β-oriented.

A preferred class of compounds that may be produced in accordance withthe methods of the invention are the 20-spiroxane compounds described inU.S. Pat. No. 4,559,332, i.e., those corresponding to Formula IA:##STR40## where: --A--A-- represents the group --CH₂ --CH₂ -- or--CH═CH--,

--B--B-- represents the group --CH₂ --CH₂ -- or an alpha- orbeta-oriented group of Formula IIIA: ##STR41## R¹ represents analpha-oriented lower alkoxycarbonyl or hydroxycarbonyl radical,

X represents two hydrogen atoms, oxo or ═S

Y¹ and Y² together represent the oxygen bridge --O--, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy,

Preferably, 20-spiroxane compounds produced by the novel methods of theinvention are those of Formula I in which Y¹ and Y² together representthe oxygen bridge --O--.

Especially preferred compounds of the formula I are those in which Xrepresents oxo.

Of compounds of the 20-spiroxane compounds of Formula IA in which Xrepresents oxo there are most especially preferred those in which Y¹together with Y² represents the oxygen bridge --O--.

As already mentioned, 17β-hydroxy-21-carboxylic acid may also be in theform of their salts. There come into consideration especially metal andammonium salts, such as alkali metal and alkaline earth metal salts, forexample sodium, calcium, magnesium and, preferably, potassium, salts,and ammonium salts derived from ammonia or a suitable, preferablyphysiologically tolerable, organic nitrogen-containing base. As basesthere come into consideration not only amines, for example loweralkylamines (such as triethylamine), hydroxy-lower alkylamines [such as2-hydroxyethylamine, di-(2-hydroxyethyl)-amine ortri-(2-hydroxyethyl)-amine], cycloalkylamines (such asdicyclohexylamine) or benzylamines (such as benzylamine andN,N'-dibenzylethylenediamine), but also nitrogen-containing heterocycliccompounds, for example those of aromatic character (such as pyridine orquinoline) or those having an at least partially saturated heterocyclicring (such as N-ethylpiperidine, morpholine, piperazine orN,N'-dimethylpiperazine).

Also included amongst preferred compounds are alkali metal salts,especially potassium salts, of compounds of the formula IA in which R¹represents alkoxycarbonyl, with X representing oxo and each of Y¹ and Y²representing hydroxy.

Especially preferred compounds of the formula I and IA are, for example,the following:

9α,11α-epoxy-7α-methoxycarbonyl-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-ene-3,21-dione

9α,11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of the compounds,

9α,11α-epoxy-6α,7α-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6β,7β-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of these compounds,

9α,11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6α,7α-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β;15β,16β-bismethylene-3-oxo-pregn-4-ene-21-carboxylicacid, and alkali metal salts, especially the potassium salt or ammoniumof each of these acids, and also a corresponding 1,2-dehydro analogue ofeach of the mentioned carboxylic acids or of a salt thereof.

9α,11α-epoxy-15β,16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy-1565β,16β-methylene-3,21-dioxo-20-spiroxa-1,4-diene-7α-carboxylicacid methyl ester,

ethyl ester and isopropyl ester,

and also 9α,11α-epoxy-3-oxo-20-spirox-4-ene-7α-carboxylic acid methylester, ethyl ester and isopropyl ester,

9α,11α-epoxy-6β,6β-methylene-20-spirox-4-en-3-one,

9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-en-3-one,

and also9α,11α-epoxy,17β-hydroxy-17α(3-hydroxy-propyl)-3-oxo-androst-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy,17β-hydroxy-17α-(3-hydroxypropyl)-6.alpha.,7α-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6.beta.,7β-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6.beta.,7β;15β,16β-bismethylene-androst-4-en-3-one,

including 17α-(3-acetoxypropyl) and 17α-(3-fromyloxypropyl) analogues ofthe mentioned androstane compounds,

and also 1,2-dehydro analogues of all the mentioned compounds of theandrost-4-en-3-one and 20-spirox-4-en-3-one series.

The chemical names of the compounds of the Formulae I and IA, and ofanalogue compounds having the same characteristic structural features,are derived according to current nomenclature in the following manner:for compounds in which Y.sup. together with Y² represents --O--, from20-spiroxane (for example a compound of the formula IA in which Xrepresents oxo and Y¹ together with Y² represents --O-- is derived from20-spiroxan-21-one); for those in which each of Y¹ and Y² representshydroxy and X represents oxo, from17β-hydroxy-17α-pregnene-21-carboxylic acid; and for those in which eachof Y¹ and Y² represents hydroxy and X represents two hydrogen atoms,from 17β-hydroxy-17α-(3-hydroxypropyl)-androstane. Since the cyclic andopen-chain forms, that is to say lactones and 17β-hydroxy-21-carboxylicacids and their salts, respectively, are so closely related to eachother that the latter may be considered merely as a hydrated form of theformer, there is to be understood hereinbefore and hereinafter, unlessspecifically stated otherwise, both in end products of the formula I andin starting materials and intermediates of analogous structure, in eachcase all the mentioned forms together.

In accordance with the invention, several separate process schemes havebeen devised for the preparation of compounds of Formula I in high yieldand at reasonable cost. Each of the synthesis schemes proceeds throughthe preparation of a series of intermediates. A number of theseintermediates are novel compounds, and the methods of preparation ofthese intermediates are novel processes.

Scheme 1

Starting with Canrenone or Related Material

One preferred process scheme for the preparation of compounds of FormulaI advantageously begins with canrenone or a related starting materialcorresponding to Formula XIII ##STR42## wherein --A--A-- represents thegroup --CHR⁴ --CHR⁵ -- or --CR⁴ ═CR⁵ --

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

--B--B-- represents the group --CHR⁶ --CHR⁷ -- or an alpha- orbeta-oriented group: ##STR43## where R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen, halo, lower alkoxy,acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ and R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

Using a bioconversion process of the type illustrated in FIGS. 1 and 2,an 11-hydroxy group of α-orientation is introduced in the compound ofFormula XIII, thereby producing a compound of Formula VIII: ##STR44##where --A--A--, --B--B--, R³, R⁸ and R⁹ are as defined above.Preferably, the compound of Formula XIII has the structure ##STR45## andthe 11α-hydroxy product has the structure ##STR46## in each of which--A--A-- represents the group --CH₂ --CH₂ -- or --CH═CH--,

--B--B-- represents the group --CH₂ --CH₂ -- or an alpha- orbeta-oriented group: ##STR47## X represents two hydrogen atoms, oxo or═S, Y¹ and Y² together represent the oxygen bridge --O--, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy,

and salts of compounds in which X represents oxo and Y² representshydroxy-, and the compound of Formula VIII produced in the reactioncorresponds to Formula VIIIA ##STR48## wherein --A--A--, --B--B--, Y¹,Y², and X are as defined in Formula XXXA. More preferably, R⁸ and R⁹together form the 20-spiroxane structure: ##STR49## --A--A-- and--B--B-- are each --CH₂ --CH₂ --, and R³ is hydrogen.

Among the preferred organisms that can be used in this hydroxylationstep are Asoergillus ochraceus NRRL 405, Aspergillus ochraceus ATCC18500, Aspergillus niger ATCC 16888 and ATCC 26693, Aspergillus nidulansATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b,Streptomyces fradiae ATCC 10745, Bacillus megaterium ATCC 14945,Pseudomonas cruciviae ATCC 13262, and Trichothecium roseum ATCC 12543.Other preferred organisms include Fusarium oxysporum f. sp. cepae ATCC11171 and Rhizopus arrhizus ATCC 11145.

Other organisms that have exhibited activity for this reaction includeAbsidia coerula ATCC 6647, Absidia glauca ATCC 22752, Actinomucorelegans ATCC 6476, Aspergillus flavipes ATCC 1030, Aspergillus fumigatusATCC 26934, Beauveria bassiana ATCC 7159 and ATCC 13144, Botryosphaeriaobtusa IMI 038560, Calonectria decora ATCC 14767, Chaetomium cochliodesATCC 10195, Corynespora cassiicola ATCC 16718, Cunninghamellablakesleeana ATCC 8688a, Cunninghamella echinulata ATCC 3655,Cunninghamella elegans ATCC 9245, Curvularia clavata ATCC 22921,Curvularia lunata ACTT 12071, Cylindrocarpon radicicola ATCC 1011,Epicoccum humicola ATCC 12722, Gongronella butleri ATCC 22822, Hypomyceschrysospermus, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC4605, Mucor griseo-cyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095,Nocardia corallina, Paecilomyces carneus ATCC 46579, Penicillum patulumATCC 24550, Pithomyces atro-olivaceus IFO 6651, Pithomyces cynodontisATCC 26150, Pycnosporium sp. ATCC 12231, Saccharopolyspora erythrae ATCC11635, Sepedonium chrysospermum ATCC 13378, Stachylidium bicolor ATCC12672, Streptomyces hyqroscopicus ATCC 27438, Streptomyces purpurascensATCC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriformeATCC 8992, Thielavia terricola ATCC 13807, and Verticillium theobromaeATCC 12474.

Additional organisms that may be expected to show activity for the11α-hydroxylation include Cephalosporium aphidicola (Phytochemistry(1996), 42(2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995),42(2), 145-150), Tieghemella orchidis (Khim.-Farm.Zh. (1986), 20(7),871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20(7),871-876), Monosporium olivaceum (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Fusarium graminearum (Acta Microbiol. Pol., Ser. B.(1973), 5(2), 103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser.B. (1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.(1987), 28(2), 197-201).

Preparatory to production scale fermentation for hydroxylation ofcanrenone or other substrates of Formula XIII, an inoculum of cells isprepared in a seed fermentation system comprising a seed fermenter, or aseries of two or more seed fermenters. A working stock spore suspensionis introduced into the first seed fermenter, together with a nutrientsolution for growth of cells. If the volume of inoculum desired orneeded for production exceeds that produced in the first seed fermenter,the inoculum volume may be progressively and geometrically amplified byprogression through the remaining fermenters in the seed fermentationtrain. Preferably, the inoculum produced in the seed fermentation systemis of sufficient volume and viable cells for achieving rapid initiationof reaction in the production fermenter, relatively short productionbatch cycles, and high production fermenter activity. Whatever thenumber of vessels in a train of seed fermenters, the second andsubsequent seed fermenters are preferably sized so that the extent ofdilution at each step in the train is essentially the same. The initialdilution of inoculum in each seed fermenter can be approximately thesame as the dilution in the production fermenter. Canrenone or otherFormula XIII substrate is charged to the production fermenter along withinoculum and nutrient solution, and the hydroxylation reaction conductedthere.

The spore suspension charged to the seed fermentation system is from avial of working stock spore suspension taken from a plurality of vialsconstituting a working stock cell bank that is stored under cryogenicconditions prior to use. The working stock cell bank is in turn derivedfrom a master stock cell bank that has been prepared in the followingmanner. A spore specimen obtained from an appropriate source, e.g.,ATCC, is initially suspended in an aqueous medium such as, for example,saline solution, nutrient solution or a surfactant solution, (e.g., anonionic surfactant such as Tween 20 at a concentration of about 0.001%by weight), and the suspension distributed among culture plates, eachplate bearing a solid nutrient mixture, typically based on anon-digestible polysaccharide such as agar, where the spores arepropagated. The solid nutrient mixture preferably contains between about0.5% and about 5% by weight glucose, between about 0.05% and about 5% byweight of a nitrogen source, e.g., peptone, between about 0.05% andabout 0.5% by weight of a phosphorus source, e.g., an ammonium or alkalimetal phosphate such as dipotassium hydrogen phosphate, between about0.25% and about 2.5% by weight yeast lysate or extract (or other aminoacid source such as meat extract or brain heart infusion), between about1% and about 2% by weight agar or other non-digestible polysaccharide.Optionally, the solid nutrient mixture may further comprise and/orcontain between about 0.1% and about 5% by weight malt extract. The pHof the solid nutrient mixture is preferably between about 5.0 and about7.0, adjusted as required by alkali metal hydroxide or orthophosphoricacid. Among useful solid growth media are the following:

1. Solid Medium #1: 1% glucose, 0.25% yeast extract, 0.3% K₂ HPO₄ and 2%agar (Bacto); pH adjusted to 6.5 with 20% NaOH.

2. Solid Medium #2: 2% peptone (Bacto), 1% yeast extract (Bacto), 2%glucose and 2% agar (Bacto); pH adjusted to 5 with 10% H₃ PO₄.

3. Solid Medium #3: 0.1% peptone (Bacto), 2% malt extract (Bacto), 2%glucose and 2% agar (Bacto); pH as is 5.3.

4. Liquid Medium: 5% blackstrap molasses, 0.5% cornsteep liquor, 0.25%glucose, 0.25% NaCl and 0.5% KH₂ PO₄, pH adjusted to 5.8.

5. Difco Mycological agar (low pH).

The number of agar plates used in the development of a master stock cellbank can be selected with a view to future demands for master stock, buttypically about 15 to about 30 plates are so prepared. After a suitableperiod of growth, e.g., 7 to 10 days, the plates are scraped in thepresence of an aqueous vehicle, typically saline or buffer, forharvesting the spores, and the resulting master stock suspension isdivided among small vials, e.g., one ml. in each of a plurality of 1.5ml vials. To prepare a working stock spore suspension for use inresearch or production fermentation operations, the contents of one ormore of these second generation master stock vials can be distributedamong and incubated on agar plates in the manner described above for thepreparation of master stock spore suspension. Where routinemanufacturing operations are contemplated, as many as 100 to 400 platesmay be used to generate second generation working stock. Each plate isscraped into a separate working stock vial, each vial typicallycontaining one ml of the inoculum produced. For permanent preservation,both the master stock suspension and the second generation productioninoculum are advantageously stored in the vapor space of a cryogenicstorage vessel containing liquid N₂ or other cryogenic liquid.

In the process illustrated in FIG. 1, aqueous growth medium is preparedwhich includes a nitrogen source such as peptone, a yeast derivative orequivalent, glucose, and a source of phosphorus such as a phosphatesalt. Spores of the microorganism are cultured in this medium in theseed fermentation system. The preferred microorganism is Aspergillusochraceus NRRL 405 (ATCC 18500). The seed stock so produced is thenintroduced into the production fermenter together with the substrate ofFormula XIII. The fermentation broth is agitated and aerated for a timesufficient for the reaction to proceed to the desired degree ofcompletion.

The medium for the seed fermenter preferably comprises an aqueousmixture which contains: between about 0.5% and about 5% by weightglucose, between about 0.05% and about 5% by weight of a nitrogensource, e.g., peptone, between about 0.05% and about 0.5% by weight of aphosphorus source, e.g., an ammonium or alkali metal phosphate such asammonium phosphate monobasic or dipotassium hydrogen phosphate, betweenabout 0.25% and about 2.5% by weight yeast lysate or extract (or otheramino acid source such as distiller's solubles), between about 1% andabout 2% by weight agar or other non-digestible polysaccharide. Aparticularly preferred seed growth medium contains about 0.05% and about5% by weight of a nitrogen source such as peptone, between about 0.25%and about 2.5% by weight of autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose, and between about 0.05% byweight and about 0.5% by weight of a phosphorus source such as ammoniumphosphate monobasic. Especially economical process operations areafforded by the use of another preferred seed culture which containsbetween about 0.5% and about 5% by weight corn steep liquor, betweenabout 0.25% and about 2.5% autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose and about 0.05% and about 0.5%by weight ammonium phosphate monobasic. Corn steep liquor is aparticularly economical source of proteins, peptides, carbohydrates,organic acids, vitamins, metal ions, trace matters and phosphates. Mashliquors from other grains may be used in place of, or in addition to,corn steep liquor. The pH of the medium is preferably adjusted withinthe range of between about 5.0 and about 7.0, e.g., by addition of analkali metal hydroxide or orthophosphoric acid. Where corn steep liquorserves as the source of nitrogen and carbon, the pH is preferablyadjusted within the range of about 6.2 to about 6.8. The mediumcomprising peptone and glucose is preferably adjusted to a pH betweenabout 5.4 and about 6.2. Among useful growth media for use in seedfermentation:

1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

2. Medium #2: 3% corn steep liquor, 1.5% yeast extract 0.3% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

Spores of the microorganism are introduced into this medium from a vialtypically containing in the neighborhood of 109 spores per ml. ofsuspension, Optimal productivity of seed generation is realized wheredilution with growth medium at the beginning of a seed culture does notreduce the spore population density below about 10⁷ per ml. Preferably,the spores are cultured in the seed fermentation system until the packedmycelial volume (PMV) in the seed fermenter is at least about 20%,preferably 35% to 45%. Since the cycle in the seed fermentation vessel(or any vessel of a plurality which comprise a seed fermentation train)depends on the initial concentration in that vessel, it may be desirableto provide two or three seed fermentation stages to accelerate theoverall process. However, it is preferable to avoid the use ofsignificantly more than three seed fermenters in series, since activitymay be compromised if seed fermentation is carried through an excessivenumber of stages. The seed culture fermentation is conducted underagitation at a temperature in the range of about 23° to about 37° C.,preferably in range of between about 24° and about 28° C.

Culture from the seed fermentation system is introduced into aproduction fermenter, together with a production growth medium. In oneembodiment of the invention, non-sterile canrenone or other substrate ofFormula XIII serves as the substrate for the reaction. Preferably, thesubstrate is added to the production fermenter in the form of a 10% to30% by weight slurry in growth medium. To increase the surface areaavailable for 11α-hydroxylation reaction, the particle size of theFormula XIII substrate is reduced by passing the substrate through anoff line micronizer prior to introduction into the fermenter. A sterilenutrient feed stock containing glucose, and a second sterile nutrientsolution containing a yeast derivative such as autolyzed yeast (orequivalent amino acid formulation based on alternative sources such asdistiller's solubles), are also separately introduced. The mediumcomprises an aqueous mixture containing: between about 0.5% and about 5%by weight glucose, between about 0.05% and about 5% by weight of anitrogen source, e.g., peptone, between about 0.05% and about 0.5% byweight of a phosphorus source, e.g., an ammonium or alkali metalphosphate such as dipotassium hydrogen phosphate, between about 0.25%and about 2.5% by weight yeast lysate or extract (or other amino acidsource such as distiller's solubles), between about 1% and about 2% byweight agar or other non-digestible polysaccharide. A particularlypreferred production growth medium contains about 0.05% and about 5% byweight of a nitrogen source such as peptone, between about 0.25% andabout 2.5% by weight of autolyzed yeast or yeast extract, between about0.5% and about 5% by weight glucose, and between about 0.05% and about0.5% by weight of a phosphorus source such as ammonium phosphatemonobasic. Another preferred production medium contains between about0.5% and about 5% by weight corn steep liquor, between about 0.25% andabout 2.5% autolyzed yeast or yeast extract, between about 0.5% andabout 5% by weight glucose and about 0.05% and about 0.5% by weightammonium phosphate monobasic. The pH of the production fermentationmedium is preferably adjusted in the manner described above for the seedfermentation medium, with the same preferred ranges for the pH ofpeptone/glucose based media and corn steep liquor based media,respectively. Useful bioconversion growth media are set forth below:

1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

2. Medium #2: 1% peptone, 1% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

3. Medium #3: 0.5% peptone, 0.5% yeast autolised (or yeast extract) and0.5% glucose; pH adjusted to 5.8 with 20% NaOH.

4. Medium #4: 3% corn steep liquor, 1.5% yeast extract 0.3% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

5. Medium #5: 2.55% corn steep liquor, 1.275% yeast extract 0.255%ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20%NaOH.

6. Medium #6: 2.1% corn steep liquor, 1.05% yeast extract 0.21% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

Non-sterile canrenone and sterile nutrient solutions are chain fed tothe production fermenter in five to twenty, preferably ten to fifteen,preferably substantially equal, portions each over the production batchcycle. Advantageously, the substrate is initially introduced in anamount sufficient to establish a concentration of between about 0.1% byweight and about 3% by weight, preferably between about 0.5% and about2% by weight, before inoculation with seed fermentation broth, thenadded periodically, conveniently every 8 to 24 hours, to a cumulativeproportion of between about 1% and about 8% by weight. Where additionalsubstrate is added every 8 hour shift, total addition may be slightlylower, e.g., 0.25% to 2.5% by weight, than in the case where substrateis added only on a daily basis. In the latter instance cumulativecanrenone addition may need to be in the range 2% to about 8% by weight.The supplemental nutrient mixture fed during the fermentation reactionis preferably a concentrate, for example, a mixture containing betweenabout 40% and about 60% by weight sterile glucose, and between about 16%and about 32% by weight sterile yeast extract or other sterile source ofyeast derivative (or other amino acid source). Since the substrate fedto the production fermenter of FIG. 1 is non-sterile, antibiotics areperiodically added to the fermentation broth to control the growth ofundesired organisms. Antibiotics such as kanamycin, tetracycline, andcefalexin can be added without disadvantageously affecting growth andbioconversion. Preferably, these are introduced into the fermentationbroth in a concentration, e.g., of between about 0.0004% and about0.002% based on the total amount of the broth, comprising, e.g., betweenabout 0.0002% and about 0.0006% kanamicyn sulfate, between about 0.0002%and about 0.006% tetracycline HCl and/or between about 0.001% and about0.003% cefalexin, again based on the total amount of broth.

Typically, the production fermentation batch cycle is in theneighborhood of 80-160 hours. Thus, portions of each of the Formula XIIIsubstrate and nutrient solutions are typically added every 2 to 10hours, preferably every 4 to 6 hours. Advantageously, an antifoam isalso incorporated in the seed fermentation system, and in the productionfermenter.

Preferably, in the process of FIG. 1, the inoculum charge to theproduction fermenter is about 0.5 to about 7%, more preferably about 1to about 2%, by volume based on the total mixture in the fermenter, andthe glucose concentration is maintained between about 0.01% and about1.0%, preferably between about 0.025% and about 0.5%, more preferablybetween about 0.05% and about 0.25% by weight with periodic additionsthat are preferably in portions of about 0.05% to about 0.25% by weight,based on the total batch charge. The fermentation temperature isconveniently controlled within a range of about 20° to about 37° C.,preferably about 24° C. to about 28° C., but it may be desirable to stepdown the temperature during the reaction, e.g., in 2° C. increments, tomaintain the packed mycelium volume (PMV) below about 60%, morepreferably below about 50%, and thereby prevent the viscosity of thefermentation broth from interfering with satisfactory mixing. If thebiomass growth extends above the liquid surface, substrate retainedwithin the biomass may be carried out of the reaction zone and becomeunavailable for the hydroxylation reaction. For productivity, it isdesirable to reach a PMV in the range of 30 to 50%, preferably 35% to45%, within the first 24 hours of the fermentation reaction, butthereafter conditions are preferably managed to control further growthwithin the limits stated above. During reaction, the pH of thefermentation medium is controlled at between about 5.0 and about 6.5,preferably between about 5.2 and about 5.8, and the fermenter isagitated at a rate of between about 400 and about 800 rpm. A dissolvedoxygen level of at least about 10% of saturation is achieved by aeratingthe batch at between about 0.2 and about 1.0 vvm, and maintaining thepressure in the head space of the fermenter at between about atmosphericand about 1.0 bar gauge, most preferably in the neighborhood of about0.7 bar gauge. Agitation rate may also been increased as necessary tomaintain minimum dissolved oxygen levels. Advantageously, the dissolvedoxygen is maintained at well above 10%, in fact as high as 50% topromote conversion of substrate. Maintaining the pH in the range of5.5±0.2 is also optimal for bioconversion. Foaming is controlled asnecessary by addition of a common antifoaming agent. After all substratehas been added, reaction is preferably continued until the molar ratioof Formula VIII product to remaining unreacted Formula XIII substrate isat least about 9 to 1. Such conversion may be achieve within the 80-160hour batch cycle indicated above

It has been found that high conversions are associated with depletion ofinitial nutrient levels below the initial charge level, and bycontrolling aeration rate and agitation rate to avoid splashing ofsubstrate out of the liquid broth. In the process of FIG. 1, thenutrient level was depleted to and then maintained at no greater thanabout 60%, preferably about 50%, of the initial charge level; while inthe processes of FIGS. 2 and 3, the nutrient level was reduced to andmaintained at no greater than about 80%, preferably about 70%, of theinitial charge level. Aeration rate is preferably no greater than onevvm, more preferably in the range of about 0.5 vvm; while agitation rateis preferably not greater than 600 rpm.

A particularly preferred process for preparation of a compound ofFormula VIII is illustrated in FIG. 2. Again the preferred microorganismis Aspergillus ochraceus NRRL 405 (ATCC 18500). In this process, growthmedium preferably comprises between about 0.5% and about 5% by weightcorn steep liquor, between about 0.5% and about 5% by weight glucose,between about 0.1% and about 3% by weight yeast extract, and betweenabout 0.05% and about 0.5% by weight ammonium phosphate. However, otherproduction growth media as described herein may also be used. The seedculture is prepared essentially in the manner described for the processof FIG. 1, using any of the seed fermentation media described herein. Asuspension of non-micronized canrenone or other Formula XIII substratein the growth medium is prepared aseptically in a blender, preferably ata relatively high concentration of between about 10% and about 30% byweight substrate. Preferably, aseptic preparation may comprisesterilization or pasteurization of the suspension after mixing. Theentire amount of sterile substrate suspension required for a productionbatch is introduced into the production fermenter at the beginning ofthe batch, or by periodical chain feeding. The particle size of thesubstrate is reduced by wet milling in an on-line shear pump whichtransfers the slurry to the production fermenter, thus obviating theneed for use of an off line micronizer. Where aseptic conditions areachieved by pasteurization rather than sterilization, the extent ofagglomeration may be insignificant, but the use of a shear pump may bedesirable to provide positive control of particle size. Sterile growthmedium and glucose solution are introduced into the production fermenteressentially in the same manner as described above. All feed componentsto the production fermenter are sterilized before introduction, so thatno antibiotics are required.

Preferably, in operation of the process of FIG. 2, the inoculum isintroduced into the production fermenter in a proportion of betweenabout 0.5% and about 7%, the fermentation temperature is between about20° and about 37° C., preferably between about 24° C. and about 28° C.,and the pH is controlled between about 4.4 and about 6.5, preferablybetween about 5.3 and about 5.5, e.g., by introduction of gaseousammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, ororthophosphoric acid. As in the process of FIG. 1, the temperature ispreferably trimmed to control growth of the biomass so that PMV does notexceed 55-60%. The initial glucose charge is preferably between about 1%and about 4% by weight, most preferably 2.5% to 3.5% by weight, but ispreferably allowed to drift below about 1.0% by weight duringfermentation. Supplemental glucose is fed periodically in portions ofbetween about 0.2% and about 1.0% by weight based on the total batchcharge, so as to maintain the glucose concentration in the fermentationzone within a range of between about 0.1% and about 1.5% by weight,preferably between about 0.25% and about 0.5% by weight. Optionally,nitrogen and phosphorus sources may be supplemented along with glucose.However, because the entire canrenone charge is made at the beginning ofthe batch cycle, the requisite supply of nitrogen and phosphorus bearingnutrients can also be introduced at that time, allowing the use of onlya glucose solution for supplementation during the reaction. The rate andnature of agitation is a significant variable. Moderately vigorousagitation promotes mass transfer between the solid substrate and theaqueous phase. However, a low shear impeller should be used to preventdegradation of the myelin of the microorganisms. Optimal agitationvelocity varies within the range of 200 to 800 rpm, depending on culturebroth viscosity, oxygen concentration, and mixing conditions as affectedby vessel, baffle and impeller configuration. Ordinarily, a preferredagitation rate is in the range of 350-600 rpm. Preferably the agitationimpeller provides a downward axially pumping function so as to assist ingood mixing of the fermented biomass The batch is preferably aerated ata rate of between about 0.3 and about 1.0 vvm, preferably 0.4 to 0.8vvm, and the pressure in the head space of the fermenter is preferablybetween about 0.5 and about 1.0 bar gauge. Temperature, agitation,aeration and back pressure are preferably controlled to maintaindissolved oxygen in the range of at least about 10% by volume during thebioconversion. Total batch cycle is typically between about 100 andabout 140 hours.

Although the principle of operation for the process of FIG. 2 is basedon early introduction of substantially the entire canrenone charge, itwill be understood that growth of the fermentation broth may be carriedout before the bulk of the canrenone is charged. Optionally, someportion of the canrenone can also be added later in the batch.Generally, however, at least about 75% of the sterile canrenone chargeshould be introduced into the transformation fermenter within 48 hoursafter initiation of fermentation. Moreover, it is desirable to introduceat least about 25% by weight canrenone at the beginning of thefermentation, or at least within the first 24 hours in order to promotegeneration of the bioconversion enzyme(s).

In a further preferred process as illustrated in FIG. 3, the entirebatch charge and nutrient solution are sterilized in the productionfermentation vessel prior to the introduction of inoculum. The nutrientsolutions that may be used, as well as the preferences among them, areessentially the same as in the process of FIG. 2. In this embodiment ofthe invention, the shearing action of the agitator impeller breaks downthe substrate agglomerates that otherwise tend to form uponsterilization. It has been found that the reaction proceedssatisfactorily if the mean particle size of the canrenone is less thanabout 200μ and at least 75% by weight of the particles are smaller than240μ. The use of a suitable impeller, e.g., a disk turbine impeller, atan adequate velocity in the range of 200 to 800 rpm, with a tip speed ofat least about 400 cm/sec., has been found to provide a shear ratesufficient to maintain such particle size characteristics despite theagglomeration that tends to occur upon sterilization within theproduction fermenter. The remaining operation of the process of FIG. 3is essentially the same as the process of FIG. 2. The processes of FIGS.2 and 3 offer several distinct advantages over the process of FIG. 1. Aparticular advantage is the amenability to use of a low cost nutrientbase such as corn steep liquor. But further advantages are realized ineliminating the need of antibiotics, simplifying feeding procedures, andallowing for batch sterilization of canrenone or other Formula XIIIsubstrate. Another particular advantage is the ability to use a simpleglucose solution rather than a complex nutrient solution forsupplementation during the reaction cycle.

In processes depicted in FIGS. 1 to 3, the product of FIG. VIII is acrystalline solid which, together with the biomass, may be separatedfrom the reaction broth by filtration or low speed centrifugation.Alternatively, the product can be extracted from the entire reactionbroth with organic solvents. Product of Formula VIII is recovered bysolvent extraction. For maximum recovery, both the liquid phase filtrateand the biomass filter or centrifuge cake are treated with extractionsolvent, but usually ≧95% of the product is associated with the biomass.Typically, hydrocarbon, ester, chlorinated hydrocarbon, and ketonesolvents may be used for extraction. A preferred solvent is a ethylacetate. Other typically suitable solvents include toluene and methylisobutyl ketone. For extraction from the liquid phase, it may beconvenient to use a volume of solvent approximately equal to the volumeof reaction solution which it contacts. To recover product the from thebiomass, the latter is suspended in the solvent, preferably in largeexcess relative to the initial charge of substrate, e.g., 50 to 100 ml.solvent per gram of initial canrenone charge, and the resultingsuspension preferably refluxed for a period of 20 minutes to severalhours to assure transfer of product to the solvent phase from recessesand pores of the biomass. Thereafter, the biomass is removed byfiltration or centrifugation, and the filter cake preferably washed withboth fresh solvent and deionized water. Aqueous and solvent washes arethen combined and the phases allowed to separate. Formula VIII productis recovered by crystallization from the solution. To maximize yield,the mycelium is contacted twice with fresh solvent. After settling toallow complete separation of the aqueous phase, product is recoveredfrom the solvent phase. Most preferably, the solvent is removed undervacuum until crystallization begins, then the concentrated extract iscooled to a temperature of 0° to 20° C., preferably about 10° to about15° C. for a time sufficient for crystal precipitation and growth,typically 8 to 12 hours.

The processes of FIG. 2, and especially that of FIG. 3, are particularlypreferred. These processes operate at low viscosity, and are amenable toclose control of process parameters such as pH, temperature anddissolved oxygen. Moreover, sterile conditions are readily preservedwithout resort to antibiotics.

The bioconversion process is exothermic, so that heat should be removed,using a jacketed fermenter or a cooling coil within the productionfermenter. Alternatively, the reaction broth may be circulated throughan external heat exchanger. Dissolved oxygen is preferably maintained ata level of at least about 5%, preferably at least about 10%, by volume,sufficient to provide energy for the reaction and assure conversion ofthe glucose to CO₂ and H₂ O, by regulating the rate of air introducedinto the reactor in response to measurement of oxygen potential in thebroth. The pH is preferably controlled at between about 4.5 and about6.5.

In each of the alternative processes for 11-hydroxylation of thesubstrate of Formula XIII, productivity is limited by mass transfer fromthe solid substrate to the aqueous phase, or the phase interface, wherereaction is understood to occur. As indicated above, productivity is notsignificantly limited by mass transfer rates so long as the particlemean particle size of the substrate is reduced to less than about 300μ,and at least 75% by weight of the particles are smaller than 240μ.However, productivity of these processes may be further enhanced incertain alternative embodiments which provide a substantial charge ofcanrenone or other Formula XIII substrate to the production fermenter inan organic solvent. According to one option, the substrate is dissolvedin a water-immiscible solvent and mixed with the aqueous growth mediuminoculum and a surfactant. Useful water-immiscible solvents include, forexample, DMF, DMSO, C₆ -C₁₂ fatty acids, C₆ -C₁₂ n-alkanes, vegetableoils, sorbitans, and aqueous surfactant solutions. Agitation of thischarge generates an emulsion reaction system having an extendedinterfacial area for mass transfer of substrate from the organic liquidphase to the reaction sites.

A second option is to initially dissolve the substrate in a watermiscible solvent such as acetone, methylethyl ketone, methanol, ethanol,or glycerol in a concentration substantially greater than its solubilityin water. By preparing the initial substrate solution at elevatedtemperature, solubility is increased, thereby further increasing theamount of solution form substrate introduced into the reactor andultimately enhancing the reactor payload. The warm substrate solution ischarged to the production fermentation reactor along with the relativelycool aqueous charge comprising growth medium and inoculum. When thesubstrate solution is mixed with the aqueous medium, precipitation ofthe substrate occurs. However, under conditions of substantialsupersaturation and moderately vigorous agitation, nucleation is favoredover crystal growth, and very fine particles of high surface area areformed. The high surface area promotes mass transfer between the liquidphase and the solid substrate Moreover, the equilibrium concentration ofsubstrate in the aqueous liquid phase is also enhanced in the presenceof a water-miscible solvent. Accordingly, productivity is promoted.

Although the microorganism may not necessarily tolerate a highconcentration of organic solvent in the aqueous phase, a concentrationof ethanol, e.g., in the range of about 3% to about 5% by weight, can beused to advantage.

A third option is to solubilize the substrate in an aqueous cyclodextrinsolution. Illustrative cyclodextrins includehydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratioof substrate:cyclodextrin can be about 1:1 to about 1:1.5,substrate:cyclodextrin. The substrate:cyclodextrin mixture can then beadded aseptically to the bioconversion reactor.

11α-Hydroxycanrenone and other products of the 11α-hydroxylation process(Formulae VIII and VIIIA) are novel compounds, which may be isolated byfiltering the reaction medium, and extracting the product from thebiomass collected on the filtration medium. Conventional organicsolvents, e.g., ethyl acetate, acetone, toluene, chlorinatedhydrocarbons, and methyl isobutyl ketone may be used for the extraction.The product of Formula VIII may then be recrystallized from an organicsolvent of the same type. The compounds of Formula VIII have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA.

Preferably, the compounds of Formula VIII correspond to Formula VIIIA inwhich --A--A-- and --B--B-- are --CH₂ --CH₂ --, R³ is hydrogen, loweralkyl or lower alkoxy, and R⁸ and R⁹ together constitute the20-spiroxane ring: ##STR50##

Further in accordance with the process of scheme 1, the compound ofFormula VIII is reacted under alkaline conditions with a source ofcyanide ion to produce an enamine compound of Formula VII ##STR51##wherein --A--A--, R³, --B--B--, R⁸ and R⁹ are as defined above. Wherethe substrate corresponds to Formula VIIIA, the product is of FormulaVIIA ##STR52## wherein --A--A--, --B--B--, R³, Y¹, Y², and X are asdefined in Formula XIII.

Cyanidation of the 11α-hydroxyl substrate of Formula VIII may be carriedout by reacting it with a cyanide ion source such as a ketonecyanohydrin, most preferably acetone cyanohydrin, in the presence of abase and a alkali metal salt, most preferably LiCl. Alternatively,cyanidation can be effected without a cyanohydrin by using an alkalimetal cyanide in the presence of an acid.

In the ketone cyanohydrin process, the reaction is conducted insolution, preferably using an aprotic polar solvent such asdimethylformamide or dimethyl sulfoxide. Formation of the enaminerequires at least two moles of cyanide ion source per mole of substrate,and preferably a slight excess of the cyanide source is used. The baseis preferably a nitrogenous base such as a dialkylamine, trialkylamine,alkanolamine, pyridine or the like. However, inorganic bases such asalkali metal carbonates or alkali metal hydroxides can also be used.Preferably, the substrate of Formula VIII is initially present in aproportion of between about 20 and about 50% by weight and the base ispresent in a proportion of between 0.5 to two equivalents per equivalentof substrate. The temperature of the reaction is not critical, butproductivity is enhanced by operation at elevated temperature. Thus, forexample, where triethylamine is used as the base, the reaction isadvantageously conducted in the range of about 80° C. to about 90° C. Atsuch temperatures, the reaction proceeds to completion in about 5 toabout 20 hours. When diisopropylethyl amine is used as the base and thereaction is conducted at 105° C., the reaction is completed at 8 hours.At the end of the reaction period, the solvent is removed under vacuumand the residual oil dissolved in water and neutralized to pH 7 withdilute acid, preferably hydrochloric. The product precipitates from thissolution, and is thereafter washed with distilled water and air dried.Liberated HCN may be stripped with an inert gas and quenched in analkaline solution. The dried precipitate is taken up in chloroform orother suitable solvent, then extracted with concentrated acid, e.g., 6NHCl The extract is neutralized to pH 7 by addition of an inorganic base,preferably an alkali metal hydroxide, and cooled to a temperature in therange of 0° C. The resulting precipitate is washed and dried, thenrecrystallized from a suitable solvent, e.g., acetone, to produce aproduct of Formula VII suitable for use in the next step of the process.

Alternatively, the reaction may be conducted in a aqueous solvent systemcomprising water-miscible organic solvent such as methanol or in abiphasic system comprising water and an organic solvent such as ethylacetate In this alternative, product may be recovered by diluting thereaction solution with water, and thereafter extracting the productusing an organic solvent such as methylene chloride or chloroform, andthen back extracting from the organic extract using concentrated mineralacid, e.g., 2N HCl. See U.S. Pat. No. 3,200,113.

According to a still further alternative, the reaction may be conductedin a water-miscible solvent such as dimethylformamide,dimethylacetamide, N-methyl, pyrolidone or dimethyl sulfoxide, afterwhich the reaction product solution is diluted with water and renderedalkaline, e.g., by addition of an alkali metal carbonate, then cooled to0° to 10° C., thereby causing the product to precipitate. Preferably,the system is quenched with an alkali metal hypohalite or other reagenteffective to prevent evolution of cyanide. After filtration and washingwith water, the precipitated product is suitable for use in the nextstep of the process.

According to a still further alternative, the enamine product of FormulaVII may be produced by reaction of a substrate of Formula VIII in thepresence of a proton source, with an excess of alkali metal cyanide,preferably NaCN, in an aqueous solvent comprising an aproticwater-miscible polar solvent such as dimethylformamide ordimethylacetamide. The proton source is preferably a mineral acid or C₁to C₅ carboxylic acid, sulfuric acid being particularly preferred.Anomalously, no discrete proton source need be added where thecyanidation reagent is commercial LiCN in DMF.

Cyanide ion is preferably charged to the reactor in a proportion ofbetween about 2.05 and about 5 molar equivalents per equivalent ofsubstrate. The mineral acid or other proton source is believed topromote addition of HCN across the 4,5 and 6,7 double bonds, and ispreferably present in a proportion of at least one mole equivalent permole equivalent substrate; but the reaction system should remain basicby maintaining an excess of alkali metal cyanide over acid present.Reaction is preferably carried out at a temperature of at least about75° C., typically 60° C. to 100° C., for a period of about 1 to about 8hours, preferably about 1.5 to about 3 hours. At the end of the reactionperiod, the reaction mixture is cooled, preferably to about roomtemperature; and the product enamine is precipitated by acidifying thereaction mixture and mixing it with cold water, preferably at about icebath temperature. Acidification is believed to close the 17-lactone,which tends to open under the basic conditions prevailing in thecyanidation. The reaction mixture is conveniently acidified using thesame acid that is present during the reaction, preferably sulfuric acid.Water is preferably added in a proportion of between about 10 and about50 mole equivalents per mole of product.

The compounds of Formula VII are novel compounds and have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula VIIcorrespond to Formula VIIA in which --A--A-- and --B--B-- are --CH₂--CH₂ --, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring: ##STR53## Most preferably thecompound of Formula VII is5'R(5'α),7'β-20'-Aminohexadecahydro-11'β-hydroxy-10'.alpha.,13'α-dimethyl-3',5-dioxospiro[furan-2(3H),17'α(5'H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile.

In the next step of the Scheme 1 synthesis, the enamine of Formula VIIis hydrolyzed to produce a diketone compound of Formula VI ##STR54##where --A--A--, R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII.Any aqueous organic or mineral acid can be used for the hydrolysis.Hydrochloric acid is preferred. To enhance productivity, awater-miscible organic solvent, such as a lower alkanol, is preferablyused as a cosolvent. The acid should be present in proportion of atleast one equivalent per equivalent of Formula VII substrate. In anaqueous system, the enamine substrate VII can be substantially convertedto the diketone of Formula VII in a period of about 5 hours at about 80°C. Operation at elevated temperature increases productivity, buttemperature is not critical. Suitable temperatures are selected based onthe volatility of the solvent system and acid.

Preferably, the enamine substrate of Formula VII corresponds to FormulaVIIA ##STR55## and the diketone product corresponds to Formula VIA##STR56## in each of which --A--A--, --B--B--, Y¹, Y² and X are asdefined in Formula VIIIA.

At the end of the reaction period, the solution is cooled to 0° and 25°C. to crystallize the product. The product crystals may berecrystallized from a suitable solvent such as isopropanol or methanolto produce a product of Formula VI suitable for use in the next step ofthe process; but recrystallization is usually not necessary. Theproducts of Formula VI are novel compounds which have substantial valueas intermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Preferably, the compounds of Formula VIcorrespond to Formula VIA in which --A--A-- and --B--B-- are --CH₂ --CH₂--, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ togetherconstitute the 20-spiroxane ring: ##STR57## Most preferably, thecompound of Formula VI is4'S(4'α),7'α-Hexadecahydro-11'α-hydroxy-10'β,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'β(2'H)-carbonitrile.

In a particularly preferred embodiment of the invention, the productenamine of Formula VII is produced from the compound of Formula VIII inthe manner described above, and converted in situ to the diketone ofFormula VI. In this embodiment of the invention, a formula VIIIsubstrate is reacted with an excess of alkali metal cyanide in anaqueous solvent containing a proton source, or optionally an excess ofketone cyanohydrin in the presence of a base and LiCl, as describedhereinabove. However, instead of cooling the reaction mixture,acidifying, and adding water in proportions calculated to causeprecipitation of the enamine, substantial cooling of the reactionmixture is preferably avoided. Water and an acid, preferably a mineralacid such as sulfuric, are indeed added to mixture at the end of thecyanidation reaction, and the proportion of acid added is sufficient toneutralize excess alkali metal cyanide, which ordinarily requiresintroduction of at least one molar equivalent acid per mole of FormulaVIII substrate, preferably between about 2 and about 5 mole equivalentsper equivalent substrate. However, the temperature is maintained at highenough, and the dilution greater enough, so that substantialprecipitation is avoided and hydrolysis of the enamine to the diketoneis allowed to proceed in the liquid phase. Thus, the process proceedswith minimum interruption and high productivity. Hydrolysis ispreferably conducted at a temperature of at least 80° C., morepreferably in the range of about 90° C. to about 100° C., for a periodof typically about 1 to about 10 hours, more preferably about 2 to about5 hours. Then the reaction mixture is cooled, preferably to atemperature of between about 0° C. and about 15° C., advantageously inan ice bath to about 5° C. to about 10° C., for precipitation of theproduct diketone of Formula VI. The solid product may be recovered, asby filtration, and impurities removed by washing with water.

In the next step of the Scheme 1 synthesis, the diketone compound ofFormula VI is reacted with a metal alkoxide to open up the ketone bridgebetween the 4 and 7 positions, cleave the bond between the carbonylgroup and the 4-carbon, and form an α-oriented alkanoyloxycarbonylsubstituent at the 7 position and eliminating cyanide at the 5-carbon.The product of this reaction is a hydroxyester compound corresponding toFormula V ##STR58## where --A--A--, R³, --B--B--, R⁸ and R⁹ are asdefined in Formula VIII, and R¹ is lower alkoxycarbonyl orhydroxycarbonyl. The metal alkoxide used in the reaction corresponds tothe formula R¹⁰ OM where M is alkali metal and R¹⁰ corresponds to thealkoxy substituent of R¹. Yields of this reaction are most satisfactorywhen the metal alkoxide is K or Na methoxide, but other lower alkoxidescan be used. A K alkoxide is particularly preferred. Phenoxides, otheraryloxides may also be used, as well as arylsulfides. The reaction isconveniently carried out in the presence of an alcohol corresponding tothe formula R¹⁰ OH where R¹⁰ is as defined above. Other conventionalsolvents may be used. Preferably, the Formula VI substrate is present ina proportion of between about 2% and about 12% by weight, morepreferably at least about 6% by weight and R¹⁰ OM is present in aproportion of between about 0.5 and about 4 moles per mole of substrate.Temperature is not critical but elevated temperature enhancesproductivity. Reaction time is typically between about 4 and about 24hours, preferably about 4 to 16 hours. Conveniently, the reaction iscarried out at atmospheric reflux temperature depending on the solventused.

In the conversion of the diketone of Formula VI to the hydroxyester ofFormula VI, by-product cyanide ion can react with the product to form5-cyanoester. Because the equilibrium is more favorable at lowconcentrations, the reaction is preferably run at rather high dilution,e.g., as high as 40:1 for reaction with Na methoxide. It has been foundthat significantly higher productivity can be realized by use of Kmethoxide rather than Na methoxide, because a dilution in the range ofabout 20:1 is generally sufficient to minimize the extent of reversecyanidation where K methoxide is the reagent.

In accordance with the invention, it has been further discovered thatthe reverse cyanidation reaction may be inhibited by taking appropriatechemical or physical measures to remove by-product cyanide ion from thereaction zone. Thus, in a further embodiment of the invention, thereaction of the diketone with alkali metal alkoxide may be carried outin the presence of an precipitating agent for cyanide ion such as, forexample, a salt comprising a cation which forms an insoluble cyanidecompound. Such salts may, for example, include zinc iodide, ferricsulfate, or essentially any halide, sulfate or other salt of an alkalineearth or transition metal that is more soluble than the correspondingcyanide. If zinc iodide is present in proportions in the range of aboutone equivalent per equivalent diketone substrate, it has been observedthat the productivity of the reaction is increased substantially ascompared to the process as conducted in the absence of an alkali metalhalide.

Even where a precipitating agent is used for removal of cyanide ion, itremains preferable to run at fairly high dilution, but by use of aprecipitating agent the solvent to diketone substrate molar ratio may bereduced significantly compared to reactions in the absence of suchagent. Recovery of the hydroxyester of Formula V can be carried outaccording to either the extractive or non-extractive proceduresdescribed below.

Preferably, the diketone substrate of Formula VI corresponds to FormulaVIA ##STR59## and the hydroxyester product corresponds to Formula VA##STR60## in each of which --A--A--, --B--B--, Y¹, Y², and X are asdefined in Formula XIII and R¹ is as defined in Formula V.

The products of Formula V are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula Vcorrespond to Formula VA in which --A--A-- and --B--B-- are --CH₂ --CH₂--, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ togetherconstitute the 20-spiroxane ring: ##STR61##

Most preferably, the compound of Formula V is Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

The compound of Formula V may be isolated by acidifying the reactionsolution, e.g., with concentrated HCl, cooling to ambient temperature,and extracting the product with an organic solvent such as methylenechloride or ethyl acetate. The extract is washed with an aqueousalkaline wash solution, dried and filtered, after which the solvent isremoved Alternatively, the reaction solution containing the product ofFormula V may be quenched with concentrated acid. The product solutionis concentrated, cooled to 0° to 25° C. and the product solid isisolated by filtration.

According to a preferred mode of recovery of the product of Formula V,methanol and HCN are removed by distillation after the conclusion of thereaction period, with water and acid being added before or during thedistillation. Addition of water before the distillation simplifiesoperations, but progressive addition during the distillation allows thevolume in the still to be maintained substantially constant. Product ofFormula V crystallizes from the still bottoms as the distillationproceeds. This mode of recovery provides a high quality crystallineproduct without extraction operations.

In accordance with a further alternative, the reaction solutioncontaining the product of Formula V may be quenched with mineral acid,e.g., 4N HCl, after which the solvent is removed by distillation.Removal of the solvent is also effective for removing residual HCN fromthe reaction product. It has been found that multiple solventextractions for purification of the compound of Formula V are notnecessary where the compound of Formula V serves as an intermediate in aprocess for the preparation of epoxymexrenone, as described herein. Infact, such extractions can often be entirely eliminated. Where solventextraction is used for product purification, it is desirable tosupplement the solvent washes with brine and caustic washes. But wherethe solvent extractions are eliminated, the brine and caustic washes aretoo. Eliminating the extractions and washes significantly enhances theproductivity of the process, without sacrificing yield or productquality, and also eliminates the need for drying of the washed solutionwith a dessicant such as sodium sulfate. The crude11α-hydroxy-7α-alkanoyloxycarbonyl product is taken up again in thesolvent for the next reaction step of the process, which is theconversion of the 11-hydroxy group to a good leaving group at the 11position thereby producing a compound of Formula IV: ##STR62## where--A--A--, R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII, R¹ isas defined in Formula V, and R² is lower arylsulfonyloxy,alkylsulfonyloxy, acyloxy or halide. Preferably, the 11α-hydroxyl isesterified by reaction with a lower alkylsulfonyl halide, an acyl halideor an acid anhydride which is added to the solution containing theintermediate product of Formula V. Lower alkylsulfonyl halides, andespecially methanesulfonyl chloride, are preferred. Alternatively, the11-α hydroxy group could be converted to a halide by reaction of asuitable reagent such as thionyl bromide, thionyl chloride, sulfurylchloride or oxalyl chloride. Other reagents for forming 11α-sulfonicacid esters include tosyl chloride, benzenesulfonyl chloride andtrifluoromethanesulfonic anhydride. The reaction is conducted in asolvent containing a hydrogen halide scavenger such as triethylamine orpyridine. Inorganic bases such as K or Na carbonate can also be used.The initial concentration of the hydroxyester of Formula V is preferablybetween about 5% and about 50% by weight. The esterification reagent ispreferably present in slight excess. Methylene chloride is aparticularly suitable solvent for the reaction, but other solvents suchas dichloroethane, pyridine, chloroform, methyl ethyl ketone,dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers,acetonitrile, toluene, and tetrahydrofuran can also be employed. Thereaction temperature is governed primarily by the volatility of thesolvent. In methylene chloride, the reaction temperature is preferablyin the range of between about -10° C. and about 10° C.

Preferably, the hydroxyester substrate of Formula V corresponds toFormula VA ##STR63## and the product corresponds to Formula IVA##STR64## in each of which --A--A--, --B--B--, Y¹, Y², and X are asdefined in Formula XIII, R¹ is lower alkanoyloxycarbonyl orhydroxycarbonyl, and R² is as defined in Formula IV.

The products of Formula IV are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula IVcorrespond to Formula VA in which --A--A-- and --B--B-- are --CH₂ --CH₂--, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ togetherconstitute the 20-spiroxane ring: ##STR65## Most preferably, thecompound of Formula IV is Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone.

If desired, the compound of Formula IV may be isolated by removal of thesolvent. Preferably, the reaction solution is first washed with anaqueous alkaline wash solution, e.g., 0.5-2N NaoH, followed by an acidwash, e.g., 0.5-2N HCl. After removal of the reaction solvent, theproduct is recrystallized, e.g., by taking the product up in methylenechloride and then adding another solvent such as ethyl ether whichlowers the solubility of the product of Formula IV, causing it toprecipitate in crystalline form.

In the recovery of the product of Formula IV, or in preparation of thereaction solution for conversion of the Formula IV intermediate to theintermediate of Formula II as is further described hereinbelow, allextractions and/or washing steps may be dispensed with if the solutionis instead treated with ion exchange resins for removal of acidic andbasic impurities. The solution is treated first with an anion exchangeresin, then with a cation exchange resin. Alternatively, the reactionsolution may first be treated with inorganic adsorbents such as basicalumina or basic silica, followed by a dilute acid wash. Basic silica orbasic alumina may typically be mixed with the reaction solution in aproportion of between about 5 and about 50 g per kg of product,preferably between about 15 and about 20 g per kg product. Whether ionexchange resins or inorganic adsorbents are used, the treatment can becarried out by simply slurrying the resin or inorganic adsorbent withthe reaction solution under agitation at ambient temperature, thenremoving the resin or inorganic adsorbent by filtration.

In an alternative and preferred embodiment of the invention, the productcompound of Formula IV is recovered in crude form as a concentratedsolution by removal of a portion of the solvent. This concentratedsolution is used directly in the following step of the process, which isremoval of the 11α-leaving group from the compound of Formula IV,thereby producing an enester of Formula II: ##STR66## where --A--A--,R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII, and R¹ is asdefined in Formula V. For purposes of this reaction, the R² substituentof the compound of Formula IV may be any leaving group the abstractionof which is effective for generating a double bond between the 9- and11-carbons. Preferably, the leaving group is a lower alkylsulfonyloxy oracyloxy substituent which is removed by reaction with an acid and analkali metal salt. Mineral acids can be used, but lower alkanoic acidsare preferred. Advantageously, the reagent for the reaction furtherincludes an alkali metal salt of the alkanoic acid utilized. It isparticularly preferred that the leaving group comprise mesyloxy and thereagent for the reaction comprise formic acid or acetic acid and analkali metal salt of one of these acids or another lower alkanoic acid.Where the leaving group is mesyloxy and the removal reagent is formicacid and potassium formate a relatively high ratio of 9,11 to11,12-olefin is observed. If free water is present during removal of theleaving group, impurities tend to be formed, particularly a 7,9-lactone##STR67## which is difficult to remove from the final product. Hence,acetic anhydride or other drying agent is used to remove the waterpresent in formic acid. The free water content of the reaction mixturebefore reaction should be maintained at a level below about 0.5%,preferably below about 0.1% by weight, as measured by Karl Fischeranalysis for water, based on total reaction solution. Although it ispreferred that the reaction mixture be kept as dry as practicable,satisfactory results have been realized with 0.3% by weight water.Preferably, the reaction charge mixture contains between about 4% andabout 50% by weight of the substrate of Formula IV in the alkanoic acid.Between about 4% and about 20% by weight of the alkali metal salt of theacid is preferably included. Where acetic anhydride is used as thedrying agent, it is preferably present in a proportion of between about0.05 moles and about 0.2 moles per mole of alkanoic acid.

It has been found that proportions of by-product 7,9-lactone and11,12-olefin in the reaction mixture is relatively low where theelimination reagent comprises a combination of trifluoroacetic acid,trifluoroacetic anhydride and potassium acetate as the reagent forelimination of the leaving group and formation of the enester(9,11-olefin). Trifluoroacetic anhydride serves as the drying agent, andshould be present in a proportion of at least about 3% by weight, morepreferably at least about 15% by weight, most preferably about 20% byweight, based on the trifluoroacetic acid eliminating reagent.

Alternatively, the 11α-leaving groups from the compound of Formula IV,may be eliminated to produce an enester of Formula II by heating asolution of Formula IV in an organic solvent such as DMSO, DMF or DMA.

Further in accordance with the invention, the compound of Formula IV isreacted initially with an alkenyl alkanoate such as isopropenyl acetatein the presence of an acid such as toluene sulfonic acid or an anhydrousmineral acid such as sulfuric acid to form the 3-enol ester: ##STR68##of the compound of Formula IV. Alternatively, the 3 enol ester can beformed by treatment with an acid anhydrides and base such as acetic acidand sodium acetate. Further alternatives include treatment with ketenein the presence of an acid to produce IV(Z). The intermediate of FormulaIV(Z) is thereafter reacted with an alkali metal formate or acetate inthe presence of formic or acetic acid to produce the Δ-9,11 enol acetateof Formula IV(Y): ##STR69## which can then be converted to the enesterof Formula II in an organic solvent, preferably an alcohol such asmethanol, by either thermal decomposition of the enol acetate orreaction thereof with an alkali metal alkoxide. The elimination reactionis highly selective to the enester of Formula II in preference to the11,12-olefin and 7,9-lactone, and this selectivity is preserved throughconversion of the enol acetate to the enone.

Preferably, the substrate of Formula IV corresponds to Formula IVA##STR70## and the enester product corresponds to Formula IIA ##STR71##in each of which --A--A--, --B--B--, Y¹, Y² ₁, and X are as defined inFormula XIII and R¹ is as defined in Formula V.

If desired, the compound of Formula II may be isolated by removing thesolvent, taking up the solid product in cold water, and extracting withan organic solvent, such as ethyl acetate. After appropriate washing anddrying steps, the product is recovered by removing the extractionsolvent. The enester is then dissolved in a solvent appropriate for theconversion to the product of Formula I. Alternatively, the enester canbe isolated by adding water to the concentrated product solution andfiltering the solid product, thereby preferentially removing the7,9-lactone. Conversion of the substrate of Formula II to the product ofFormula IA may be conducted in the manner described in U.S. Pat. No.4,559,332 which is expressly incorporated herein by reference, or morepreferably by the novel reaction using a haloacetamide promoter asdescribed below.

In another embodiment of the invention, the hydroxyester of Formula Vmay be converted to the enester of Formula II without isolation of theintermediate compound of Formula IV. In this method, the hydroxyester istaken up in a an organic solvent, such as methylene chloride; and eitheran acylating agent, erg., methanesulfonyl chloride, or halogenatingreagent, e.g., sulfuryl chloride, is added to the solution. The mixtureis agitated and, where halogenation is involved, an HCl scavenger suchas imidazole is added. Mixing of base with the solution is highlyexothermic, and should therefore be conducted at a controlled rate withfull cooling. After the base addition, the resulting mixture is warmedto moderate temperature, e.g., 0° C. to room temperature or slightlyabove, and reacted for a period of typically 1 to 4 hours, Afterreaction is complete, the solvent is stripped, preferably under highvacuum (e.g., 24" to 28" Hg) conditions at -10° to +15° C., morepreferably about 0° to about 50° C., to concentrate the solution andremove excess base. The substrate is then redissolved in an organicsolvent, preferably a halogenated solvent such as methylene chloride forconversion to the enester.

The leaving group elimination reagent is preferably prepared by mixingan organic acid, an organic acid salt and a drying agent, preferablyformic acid, alkali metal formate and acetic anhydride, respectively, ina dry reactor. Addition of acetic anhydride is exothermic and results inrelease of CO, so the addition rate must be controlled accordingly. Topromote the removal of water, the temperature of this reaction ispreferably maintained in the range of 60° to 90° C., most preferablyabout 65° to about 75° C. This reagent is then added to the productsolution of the compound of Formula IV to effect the eliminationreaction. After 4-8 hours, the reaction mixture is preferably heated toa temperature of at least about 85° C., but not above about 95° C. untilall volatile distillate has been removed, and then for an additionalperiod to complete the reaction, typically about 1 to 4 hours. Thereaction mixture is cooled, and after recovery by standard extractiontechniques, the enester may be recovered as desired by evaporating thesolvent.

It has further been found that the enester of Formula II may berecovered from the reaction solution by an alternative procedure whichavoids the need for extraction steps following the elimination reaction,thereby providing savings in cost, improvement in yield and/orimprovement in productivity. In this process, the enester product isprecipitated by dilution of the reaction mixture with water afterremoval of formic acid. The product is then isolated by filtration. Noextractions are required.

According to a further alternative for conversion of the hydroxyester ofFormula V to the enester of Formula II without isolation of the compoundof Formula IV, the 11α-hydroxy group of the Formula V hydroxyester isreplaced by halogen, and the Formula II enester is then formed in situby thermal dehydro halogenation. Replacement of the hydroxy group byhalogen is effected by reaction with sulfuryl halide, preferablysulfuryl chloride, in the cold in the presence of a hydrogen halidescavenger such as imidazole. The hydroxyester is dissolved in a solventsuch as tetrahydrofuran and cooled to 0° C. to -70° C. The sulfurylhalide is added and the reaction mixture is warmed to moderatetemperature, e.g., room temperature, for a time sufficient to completethe elimination reaction, typically 1 to 4 hours. The process of thisembodiment not only combines two steps into one, but eliminates the useof: a halogenated reaction solvent; an acid (such as acetic); and adrying reagent (acetic anhydride or sodium sulfate). Moreover, thereaction does not require refluxing conditions, and avoids thegeneration of by-product CO which results when acetic acid is used as adrying reagent.

In accordance with a particularly preferred embodiment of the invention,the diketone compound of Formula VI can be converted to epoxymexrenoneor other compound of Formula I without isolating any intermediate inpurified form. In accordance with this preferred process, the reactionsolution containing the hydroxyester is quenched with a strong acidsolution, cooled to ambient temperature and then extracted with anappropriate extraction solvent. Advantageously, an aqueous solution ofinorganic salt, e.g., 10% by weight saline solution, is added to thereaction mixture prior to the extraction. The extract is washed anddried by azeotropic distillation for removal of the methanol solventremaining from the ketone cleavage reaction.

The resulting concentrated solution containing between about 5% andabout 50% by weight compound of Formula V is then contacted in the coldwith an acylating or alkylsulfonylating reagent to form the sulfonicester or dicarboxylic acid ester. After the alkylsulfonation orcarboxylation reaction is complete the reaction solution is passed overan acidic and then a basic exchange resin column for the removal ofbasic and acidic impurities. After each pass, the column is washed withan appropriate solvent, erg., methylene chloride, for the recovery ofresidual sulfonic or dicarboxylic ester therefrom. The combined eluateand wash fractions are combined and reduced, preferably under vacuum, toproduce a concentrated solution containing the sulfonic ester ordicarboxylic ester of Formula IV. This concentrated solution is thencontacted with a dry reagent comprising an agent effect for removal ofthe 11α-ester leaving group and abstraction of hydrogen to form a 9,11double bond. Preferably, the reagent for removal of the leaving groupcomprises the formic acid/alkali metal formate/acetic anhydride dryreagent solution described above. After reaction is complete, thereaction mixture is cooled and formic acid and/or other volatilecomponents are removed under vacuum. The residue is cooled to ambienttemperature, subjected to appropriate washing steps, and then dried togive a concentrated solution containing the enester of Formula II. Thisenester may then be converted to epoxymexrenone or other compound ofFormula I using the method described herein, or in U.S. Pat. No.4,559,332.

In an especially preferred embodiment of the invention, the solvent isremoved from the reaction solution under vacuum, and the product ofFormula IV is partitioned between water and an appropriate organicsolvent, e.g., ethyl acetate. The aqueous layer is then back extractedwith the organic solvent, and the back extract washed with an alkalinesolution, preferably a solution of an alkali metal hydroxide containingan alkali metal halide. The organic phase is concentrated, preferablyunder vacuum, to yield the enester product of Formula II. The product ofFormula II may then be taken up in an organic solvent, e.g., methylenechloride, and further reacted in the manner described in the '332 patentto produce the product of Formula I.

Where trihaloacetonitrile is used in the epoxidation reaction, it hasbeen found that the selection of solvent is important, with halogenatedsolvents being highly preferred, and methylene chloride being especiallypreferred. Solvents such as dichloroethane and chlorobenzene givereasonably satisfactory yields, but yields are generally better in amethylene chloride reaction medium. Solvents such as acetonitrile andethyl acetate generally give poor yields, while reaction in solventssuch as methanol or water/tetrahydrofuran give little of the desiredproduct

Further in accordance with the present invention, it has been discoveredthat numerous improvements in the synthesis of epoxymexrenone can berealized by use of a trihaloacetamide rather than a trihaloacetonitrileas a peroxide activator for the epoxidation reaction In accordance witha particularly preferred process, the epoxidation is carried out byreaction of the substrate of Formula IIA with hydrogen peroxide in thepresence of trichloroacetamide and an appropriate buffer. Preferably,the reaction is conducted in a pH in the range of about 3 to about 7,most preferably between about 5 and about 7. However, despite theseconsiderations, successful reaction has been realized outside thepreferred pH ranges.

Especially favorable results are obtained with a buffer comprisingdipotassium hydrogen phosphate, and/or with a buffer comprising acombination of dipotassium hydrogenphosphate and potassium dihydrogenphosphate in relative proportions of between about 1:4 and about 2:1,most preferably in the range of about 2:3. Borate buffers can also beused, but generally give slower conversions than dipotassium phosphateor K₂ HPO₄ or K₂ HPO₄ /KH₂ PO₄ mixtures. Whatever the makeup of thebuffer, it should provide a pH in the range indicated above. Aside fromthe overall composition of the buffer or the precise pH it may impart,it has been observed that the reaction proceeds much more effectively ifat least a portion of the buffer is comprised of dibasichydrogenphosphate ion. It is believed that this ion may participateessentially as a homogeneous catalyst in the formation of an adduct orcomplex comprising the promoter and hydroperoxide ion, the generation ofwhich may in turn be essential to the overall epoxidation reactionmechanism. Thus, the quantitative requirement for dibasichydrogenphosphate (preferably from K₂ HPO₄) may be only a smallcatalytic concentration. Generally, it is preferred that HPO₄ be presentin a proportion of at least about 0.1 equivalents, e.g., between about0.1 and about 0.3 equivalents, per equivalent substrate.

The reaction is carried out in a suitable solvent, preferably methylenechloride, but alternatively other halogenated solvents such aschlorobenzene or dichloroethane can be used. Toluene and mixtures oftoluene and acetonitrile have also been found satisfactory. Withoutcommitting to a particular theory, it is posited that the reactionproceeds most effectively in a two phase system in which a hydroperoxideintermediate is formed and distributes to the organic phase of low watercontent, and reacts with the substrate in the organic phase. Thus thepreferred solvents are those in which water solubility is low. Effectiverecovery from toluene is promoted by inclusion of another solvent suchas acetonitrile.

In the conversion of substrates of Formula II to products of Formula I,toluene provides a process advantage since the substrates are freelysoluble in toluene and the products are not. Thus, the productprecipitates during the reaction when conversions reach the 40-50%range, producing a three phase mixture from which the product can beconveniently separated by filtration. Methanol, ethyl acetate,acetonitrile alone, THF and THF/water have not proved as to be aseffective as the halogenated solvents or toluene in carrying out theconversion of this step of the process.

While trichloroacetamide is a highly preferred reagent, othertrihaloacetamides such as trifluoroacetamide can also be used.Trihalomethylbenzamide, and other compounds having an arylene moietybetween the electron withdrawing trihalomethyl group and the carbonyl ofthe amide, may also be useful. 3,3,3-Trihalopropionamides may also beused, but with less favorable results Generically, the peroxideactivator may correspond to the formula:

    R.sup.o C(O)NH.sub.2

where R^(o) is a group having an electron withdrawing strength (asmeasured by sigma constant) at least as high as that of themonochloromethyl group. More particularly, the peroxide activator maycorrespond to the formula: ##STR72## where X¹, X², and X³ areindependently selected from among halo, hydrogen, alkyl, haloalkyl andcyano and cyanoalkyl, and R^(p) is selected from among arylene and--(CX⁴ X⁵)_(n) --, where n is 0 or 1, at least one of X¹, X², X³, X⁴ andX⁵ being halo or perhaloalkyl. Where any of X¹, X², X³, X⁴ or X⁵ is nothalo, it is preferably haloalkyl, most preferably perhaloalkyl.Particularly preferred activators include those in which n is 0 and atleast two of X¹, X² and X³ are halo; or in which all of X¹, X², X³, X⁴and X⁵ are halo or perhaloalkyl. Each of X¹, X² X³, X⁴ and X⁵ ispreferably Cl or F, most preferably Cl, though mixed halides may also besuitable, as may perchloralkyl or perbromoalkyl and combinationsthereof.

Preferably, the peroxide activator is present in a proportion of atleast about 1 equivalents, more preferably between about 1.5 and about 2equivalents, per equivalent of substrate initially present. Hydrogenperoxide should be charged to the reaction in at least modest excess, oradded progressively as the epoxidation reaction proceeds. Although thereaction consumes only one to two equivalents of hydrogen peroxide permole of substrate, hydrogen peroxide is preferably charged insubstantial excess relative to substrate and activator initiallypresent. Without limiting the invention to a particular theory, it isbelieved that the reaction mechanism involves formation of an adduct ofthe activator and OOH⁻, that the formation of this reaction isreversible with the equilibrium favoring the reverse reaction, and thata substantial initial excess of hydrogen peroxide is therefore necessaryin order to drive the reaction in the forward direction. Temperature ofthe reaction is not narrowly critical, and may be effectively carriedout within the range of 0° to 100° C. The optimum temperature depends onthe selection of solvent. Generally, the preferred temperature isbetween about 20° C. and 30° C., but in certain solvents, e.g., toluenethe reaction may be advantageously conducted in the range of 60°-70° C.At 25° C., reaction typically requires less than 10 hours, typically 3to 6 hours. If needed additional activator and hydrogen peroxide may beadded at the end of the reaction cycle to achieve complete conversion ofthe substrate.

At the end of the reaction cycle, the aqueous phase is removed, theorganic reaction solution is preferably washed for removal of watersoluble impurities, after which the product may be recovered by removalof the solvent. Before removal of solvent, the reaction solution shouldbe washed, at with least a mild to moderately alkaline wash, e.g.,sodium carbonate. Preferably, the reaction mixture is washedsuccessively with: a mild reducing solution such as a weak (e.g. 3% byweight) solution of sodium sulfite in water; an alkaline solution, e.g.,NaOH or KOH (preferably about 0.5N); an acid solution such as HCl(preferably about 1N); and a final neutral wash comprising water orbrine, preferably saturated brine to minimize product losses. Prior toremoval of the reaction solvent, another solvent such as an organicsolvent, preferably ethanol may be advantageously added, so that theproduct may be recovered by crystallization after distillation forremoval of the more volatile reaction solvent.

It should be understood that the novel epoxidation method utilizingtrichloroacetamide or other novel peroxide activator has applicationwell beyond the various schemes for the preparation of epoxymexrenone,and in fact may be used for the formation of epoxides across olefinicdouble bonds in a wide variety of substrates subject to reaction in theliquid phase. The reaction is particularly effective in unsaturatedcompounds in which the olefinic carbons are tetrasubstituted andtrisubstituted, i.e., R^(a) R^(b) C═CR^(c) R^(d) and R^(a) R^(b)C═CR^(c) RH where R^(a) to R^(d) represent substituents other thanhydrogen. The reaction proceeds most rapidly and completely where thesubstrate is a cyclic compound with a trisubstituted double, or either acyclic or acyclic compound with tetrasubstituted double bonds. Exemplarysubstrates for this reaction include Δ-9,11-canrenone, and ##STR73##

Because the reaction proceeds more rapidly and completely withtrisubstituted and tetrasubstituted double bonds, it is especiallyeffective for selective epoxidation across such double bonds incompounds that may include other double bonds where the olefinic carbonsare monosubstituted, or even disubstituted.

It should be further understood that the reaction may be used toadvantage in the epoxidation of monosubstituted or even disubstituteddouble bonds, such as the 11,12-olefin in various steroid substrates.However, because it preferentially epoxidizes the more highlysubstituted double bonds, e.g., the 9,11-olefin, with high selectivity,the process of this invention is especially effective for achieving highyields and productivity in the epoxidation steps of the various reactionschemes described elsewhere herein.

The improved process has been shown to be particularly advantageousapplication to the preparation of: ##STR74## by epoxidation of:##STR75##

Multiple advantages have been demonstrated for the process of theinvention in which trichloroacetamide is used in place oftrichloroacetonitrile as the oxygen transfer reagent for the epoxidationreaction. The trichloroacetamide reagent system provides tightregiocontrol for epoxidation across trisubstituted double withdisubstituted and α,β-keto olefins in the same molecular structure.Thus, reaction yield, product profile and final purity are substantiallyenhanced. It has further been discovered that the substantial excessoxygen generation observed with the use of trihaloacetonitrile is notexperienced with trichloroacetamide, imparting improved safety to theepoxidation process. Further in contrast to the trichloroacetonitrilepromoted reaction, the trichloroacetamide reaction exhibits minimumexothermic effects, thus facilitating control of the thermal profile ofthe reaction. Agitation effects are observed to be minimal and reactorperformance more consistent, a further advantage over thetrichloroacetonitrile process The reaction is more amenable to scaleupthan the trichloroacetonitrile promoted reaction. Product isolation andpurification is simple, there is no observable Bayer-Villager oxidationof carbonyl function (peroxide promoted conversion of ketone to ester)as experienced, e.g., using m-chloroperoxybenzoic acid or other peracidsand the reagent is inexpensive, readily available, and easily handled.

The novel epoxidation method of the invention is highly useful as theconcluding step of the synthesis of Scheme 1. In a particularlypreferred embodiment, the overall process of Scheme 1 proceeds asfollows: ##STR76##

Scheme 2

The second of novel reaction schemes (Scheme 2) of this invention startswith canrenone or other substrate corresponding to Formula XIII##STR77## where --A--A--, R³, --B--B--, R⁸ and R⁹ are as defined inFormula VIII. In the first step of this process, the substrate ofFormula XIII is converted to a product of Formula XII ##STR78## using acyanidation reaction scheme substantially the same as that describedabove for conversion of the substrate of Formula VIII to theintermediate of Formula VII. Preferably, the substrate of Formula XIIIcorresponds to Formula XIIIA ##STR79## and the enamine productcorresponds to Formula XIIA ##STR80## in each of which --A--A--,--B--B--, Y¹, Y², and X are as defined in Formula XIII.

In the second step of scheme 2, the enamine of Formula XII is hydrolyzedto an intermediate diketone product of Formula XI ##STR81## where--A--A--, R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII, usinga reaction scheme substantially the same as that described above forconversion of the substrate of Formula VIII to the intermediate ofFormula VII. Preferably, the substrate of Formula XII corresponds toFormula XIIA ##STR82## and the diketone product corresponds to FormulaXIA ##STR83## in each of which --A--A--, --B--B--, Y¹, Y², and X are asdefined in Formula VIIIA.

Further in accordance with reaction scheme 2, the diketone of Formula XIis reacted with an alkali metal alkoxide to form mexrenone or otherproduct corresponding to Formula X, ##STR84## in each of which --A--A--,R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII. R¹ is as definedin Formula V. The process is carried out using substantially the samereaction scheme that is described above for the conversion of thecompounds of Formula VI to those of Formula V. Preferably, the substrateof Formula XI corresponds to Formula XIA ##STR85## and the intermediateproduct corresponds to Formula XA ##STR86## in each of which --A--A--,--B--B--, Y¹, Y², and X are as defined in Formula XIII. R¹ is as definedin Formula V.

Canrenone and other compounds of Formula X are next 9α-hydroxylated by anovel bioconversion process to yield products of Formula IX ##STR87##where --A--A--, R³, --B--B--, R⁸ and R⁹ are as defined in Formula VIII,and R¹ is as defined in Formula V. Among the organisms that can be usedin this hydroxylation step are Nocardia conicruria ATCC 31548, Nocardiaaurentia ATCC 12674, Corynespora cassiicola ATCC 16718, Streptomyceshyqroscopicus ATCC 27438, Mortierella isabellina ATCC 42613, Beauvriabassiana ATCC 7519, Penicillum purpurogenum ATCC 46581, Hypomyceschrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992,Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Trichothecium roseum ATCC 12543,Epicoccum humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635,Beauvria bassiana ATCC 13144, Arthrobacter simplex, Bacteriumcyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, Nocardiaaurentia ATCC 12674, Nocardia canicruria, Norcardia restrictus ATCC14887, Pseudomonas testosteroni ATCC 11996, Rhodococcus equi ATCC 21329,Mycobacterium fortuitum ATCC-6842, and Rhodococcus rhodochrous ATCC19150. The reaction is carried out substantially in the manner describedabove in connection with FIGS. 1 and 2. The process of FIG. 1 isparticularly preferred.

Growth media useful in the bioconversions preferably contain betweenabout 0.05% and about 5% by weight available nitrogen; between about0.5% and about 5% by weight glucose; between about 0.25% and about 2.5%by weight of a yeast derivative; and between about 0.05%, and about 0.5%by weight available phosphorus. Particularly preferred growth mediainclude the following:

soybean meal: between about 0.5% and about 3% by weight glucose; betweenabout 0.1% and about 1% by weight soybean meal; between about 0.05% andabout 0.5% by weight alkali metal halide; between about 0.05% and about560.5% by weight of a yeast derivative such as autolyzed yeast or yeastextract; between about 0.05% and about 0.5% by weight of a phosphatesalt such as K₂ HPO₄ ; pH=7;

peptone-yeast extract-glucose: between about 0.2% and about 2% by weightpeptone; between about 0.05% and about 0.5% by weight yeast extract; andbetween about 2% and about 5% by weight glucose;

Mueller-Hinton: between about 10% and about 40% by weight beef infusion;between about 0.35% and about 8.75% by weight casamino acids; betweenabout 0.15% and about 0.7% by weight starch.

Fungi can be grown in soybean meal or peptone nutrients, whileactinomycetes and eubacteria can be grown in soybean meal (plus 0.5% to1% by weight carboxylic acid salt such as Na formate forbiotransformations) or in Mueller-Hinton broth.

The production of 11β-hydroxymexrenone from mexrenone by fermentation isdiscussed in Example 19.

The products of Formula IX are novel compounds, which may be separatedby filtration, washed with a suitable organic solvent, e.g., ethylacetate, and recrystallized from the same or a similar solvent. Theyhave substantial value as intermediates for the preparation of compoundsof Formula I, and especially of Formula IA. Preferably, the compounds ofFormula IX correspond to Formula IXA in which --A--A-- and --B--B-- are--CH₂ --CH₂ --, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ andR⁹ together constitute the 20-spiroxane ring: ##STR88##

In the next step of synthesis scheme 2, the product of Formula IX isreacted with a dehydration reagent to produce a compound of Formula II##STR89## wherein --A--A--, R³, --B--B--, R⁸ and R⁹ are as defined inFormula VIII, and R¹ is as defined in Formula V. Where the substratecorresponds to Formula IXA, the product is of Formula IIA ##STR90## ineach of which --A--A--, --B--B--, Y¹, Y², and X are as defined inFormula XIII and R¹ is as defined in Formula V.

In the final step of this synthesis scheme, the product of Formula II isconverted to that of Formula I by epoxidation in accordance with themethod described in U.S. Pat. No. 4,559,332; or preferably by the novelepoxidation method of the invention as described hereinabove.

In a particularly preferred embodiment, the overall process of Scheme 2proceeds as follows: ##STR91##

Scheme 3

The synthesis in this case begins with a substrate corresponding toFormula XX ##STR92## where --A--A-- and R³ are as defined in FormulaVIII, --B--B-- is as defined in Formula VIII except that neither R⁶ norR⁷ is part of a ring fused to the D ring at the 16,17 positions, and R²⁶is lower alkyl, preferably methyl. Reaction of the substrate of FormulaXX with a sulfonium ylide produces the epoxide intermediatecorresponding to Formula XIX ##STR93## wherein --A--A--, R³, --B--B--,and R²⁶ are as defined in Formula XX.

In the next step of synthesis scheme 3, the intermediate of Formula XIXis converted to a further intermediate of Formula XVIII ##STR94##wherein --A--A--, R³, and --B--B-- are as defined in Formula XX. In thisstep, Formula XIX substrate is converted to Formula XVIII intermediateby reaction with NaCH(COOEt)₂ in the presence of a base in a solvent.Exposure of the compound of Formula XVIII to heat water and an alkalihalide produces a decarboxylated intermediate compound corresponding toFormula XVII ##STR95## wherein --A--A--, R³, and --B--B-- are as definedin Formula XX. The process for conversion of the compound of Formula XXto the compound of Formula XVII corresponds essentially to thatdescribed in U.S. Pat. Nos. 3,897,417, 3,413,288 and 3,300,489, whichare expressly incorporated herein by reference. While the substratesdiffer, the reagents, mechanisms and conditions for introduction of the17-spirolactone moiety are essentially the same.

Reaction of the intermediate of Formula XVII with a dehydrogenationreagent yields the further intermediate of Formula XVI. ##STR96## where--A--A--, R³ and --B--B-- are as defined above.

Typically useful dehydrogenation reagents includedichlorodicyanobenzoquinone (DDQ) and chloranil(2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the dehydrogenationcould be achieved by a sequential halogenation at the carbon-6 followedby dehydrohalogenation reaction.

The intermediate of Formula XVI is next converted to the enamine ofFormula XV ##STR97## wherein --A--A--, R³ and --B--B-- are as defined inFormula XX. Conversion is by cyanidation essentially in the mannerdescribed above for the conversion of the 11α-hydroxy compound ofFormula VIII to the enamine of Formula VII. Typically, the cyanide ionsource may be an alkali metal cyanide. The base is preferablypyrrolidine and/or tetramethylguanidine. A methanol solvent may be used.

The products of Formula XV are novel compounds, which may be isolated bychromatography. These and other novel compounds of Formula AXV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula AXVcorrespond to the structure ##STR98## where --A--A--, --B--B--, R³, R⁸and R⁹ are as defined above. In the most preferred compounds of FormulaXV, and --A--A-- and --B--B-- are --CH₂ --CH₂ --.

In accordance with the hydrolysis described above for producing thediketone compounds of Formula VI, the enamines of Formula XV may beconverted to the diketones of Formula XIV ##STR99## wherein --A--A--,R³, and --B--B-- are as defined in Formula XX. Particularly preferredfor the synthesis of epoxymexrenone are those compounds of Formula XIVwhich also fall within the scope of Formula VIA.

The products of Formula XIV are novel compounds, which may be isolatedby precipitation. These and other novel compounds of Formula AXIV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula AXIVcorrespond to the structure ##STR100## where --A--A--, --B--B--, R³, R⁸and R⁹ are as defined above. In the most preferred compounds of FormulaAXIV and XIV, --A--A-- and --B--B-- are --CH₂ --CH₂ --.

The compounds of Formula XIV are further converted to compounds ofFormula XXXI using essentially the process described above forconverting the diketone of Formula VI to the hydroxyester of Formula V.In this instance, it is necessary to isolate the intermediate XXXI##STR101## before further conversion to a product of Formula XXXII##STR102## wherein --A--A-- and --B--B-- are as defined in Formula XX.Preferred compounds of Formula XXXI are those which fall within FormulaIIA. The compounds of Formula XXXI are converted to compounds of FormulaXXXII using the method described hereinabove or in U.S. Pat. No.4,559,332. In a particularly preferred embodiment, the overall processof Scheme 3 proceeds as follows: ##STR103##

Scheme 4

The first three steps of Scheme 4 are the same as those of Scheme 3,i.e., preparation of an intermediate of Formula XVII starting with acompound corresponding to Formula XX.

Thereafter, the intermediate of Formula XVII is epoxidized, for example,using the process of U.S. Pat. No. 4,559,332 to produce the compound ofFormula XXIV ##STR104## wherein --A--A--, R³, and --B--B-- are asdefined in Formula XX. However, in a particularly preferred embodimentof the invention, the substrate of Formula XVII is epoxidized across the9,11-double bond using an oxidation reagent comprising an amide typeperoxide activator, most preferably trichloroacetamide, according to theprocess as described above in Scheme 1 for the conversion of the enesterof Formula II to the product of Formula I. The conditions andproportions of reagents for this reaction are substantially as describedfor the conversion of the Formula II enester to epoxymexrenone.

It has been found that the epoxidation of the substrate of Formula XVIIcan also be effected in very good yield using a peracid such as, forexample, m-chloroperoxybenzoic acid. However, the trichloroacetamidereagent provides superior results in minimizing the formation ofBayer-Villager oxidation by-product. The latter by-product can beremoved, but this requires trituration from a solvent such as ethylacetate, followed by crystallization from another solvent such asmethylene chloride. The epoxy compound of Formula XXIV is dehydrogenatedto produce a double bond between the 6- and 7-carbons by reaction with adehydrogenation (oxidizing) agent such as DDQ or chloranil, or using thebromination/dehydrobromination (or otherhalogenation/dehydrohalogenation) sequence, to produce another novelintermediate of Formula XXIII ##STR105## wherein --A--A--, and --B--B--are as defined in Formula XX. Particularly preferred compounds ofFormula XXIII are those in which --A--A-- and --B--B-- are as defined inFormula XIII.

While direct oxidation is effective for the formation of the product ofFormula XXIII, the yields are generally low. Preferably, therefore, theoxidation is carried out in two steps, first halogenating the substrateof Formula XXIV at the C-6 position, then dehydrohalogenating to the6,7-olefin. Halogenation is preferably effected with an N-halo organicreagent such as, for example, N-bromosuccinamide. Bromination is carriedout in a suitable solvent such as, for example, acetonitrile, in thepresence of halogenation promoter such as benzoyl peroxide. The reactionproceeds effectively at a temperature in the range of about 50° to about100° C., conveniently at atmospheric reflux temperature in a solventsuch as carbon tetrachloride, acetonitrile or mixture thereof. However,reaction from 4 to 10 hours is typically required for completion of thereaction. The reaction solvent is stripped off, and the residue taken upa water-immiscible solvent, e.g., ethyl acetate. The resulting solutionis washed sequentially with a mild alkaline solution (such as an alkalimetal bicarbonate) and water, or preferably saturated brine to minimizeproduct losses, after which the solvent is stripped and a the residuetaken up in another solvent (such as dimethylformamide) that is suitablefor the dehydrohalogenation reaction.

A suitable dehydrohalogenation reagent, e.g.,1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the solution, alongwith an alkali metal halide such as LiBr, the solution heated to asuitable reaction temperature, e.g., 60° to 80° C., and reactioncontinued for several hours, typically 4 to 15 hours, to complete thedehydrobromination. Additional dehydrobromination reagent may be addedas necessary during the reaction cycle, to drive the reaction tocompletion. The product of Formula XXIII may then be recovered, e.g., byadding water to precipitate the product which is then separated byfiltration and preferably washed with additional amounts of water. Theproduct is preferably recrystallized, for example fromdimethylformamide.

The products of Formula XXIII, such as 9,11-epoxycanrenone, are novelcompounds, which may be isolated by extraction/crystallization. Theyhave substantial value as intermediates for the preparation of compoundsof Formula I, and especially of Formula IA. For example, they may beused as substrates for the preparation of compounds of Formula XXII. Inthe most preferred compounds of Formula XXIII, and --A--A-- and --B--B--are --CH₂ --CH₂ --.

Using substantially the process described above for the preparation ofcompounds of Formula VII, the compounds of Formula XXIII are reactedwith cyanide ion to produce novel epoxyenamine compounds correspondingto Formula XXII ##STR106## wherein --A--A--, R³, and --B--B-- are asdefined in Formula XX. Particularly preferred compounds of Formula XXIIare those in which --A--A-- and --B--B-- are as defined in Formula XIII.

The products of Formula XXII are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. In the most preferred compounds of FormulaXXII, and --A--A-- and --B--B-- are --CH₂ --CH₂ --.

Using substantially the process described above for preparation ofcompounds of Formula VI, the epoxyenamine compounds of Formula XXII areconverted to novel epoxydiketone compounds of Formula XXI.

The products of Formula XXI are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXI are those in which --A--A-- and --B--B-- are as defined in FormulaXIII. In the most preferred compounds of Formula XXI, and --A--A-- and--B--B-- are --CH₂ --CH₂ --.

Compounds of Formula XXI are converted to compounds of Formula XXXIIusing the epoxidation process described hereinabove or the process ofU.S. Pat. No. 4,559,332. In a particularly preferred embodiment, theoverall process of Scheme 4 proceeds as follows: ##STR107##

Scheme 5

The process of scheme 5 begins with a substrate corresponding to FormulaXXIX ##STR108## wherein --A--A--, and --B--B-- are as defined in FormulaXX. This substrate is converted to a product of Formula XXVIII##STR109## by reaction with trimethylorthoformate. wherein --A--A--, R³,and --B--B-- are as defined in Formula XX.

Following the formation of Formula XXVIII, the compounds of Formula XXIXare converted to compounds of Formula XXVII using the method describedabove for conversion of the substrate of Formula XX to Formula XVII.Compounds of Formula XXVII have the structure: ##STR110## wherein--A--A--, and --B--B-- are as defined in Formula XX, and R^(x) is any ofthe common hydroxyl protecting groups.

Using the method described above for the preparation of compounds ofFormula XVI, compounds of Formula XXVII are oxidized to yield novelcompounds corresponding to Formula XXVI ##STR111## wherein --A--A--, and--B--B-- are as defined in Formula XX. Particularly preferred compoundsof Formulae XXIX, XXVIII, XXVII and XXVI are those in which --A--A-- and--B--B-- are as defined in Formula XIII.

The products of Formula XXVI are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXVI are those in which --A--A-- and --B--B-- are as defined in FormulaXIII. In the most preferred compounds of Formula XXVI, and --A--A-- and--B--B-- are --CH₂ --CH₂ --.

Using the method defined above for cyanidation of compounds of FormulaVIII, the novel intermediates of Formula XXVI are converted to the novel9-hydroxyenamine intermediates of Formula XXV ##STR112## wherein--A--A--, R³, and --B--B-- are as defined in Formula XX.

The products of Formula XXV are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXV are those in which --A--A-- and --B--B-- are as defined in FormulaXIII. In the most preferred compounds of Formula XXVI, and --A--A-- and--B--B-- are --CH₂ --CH₂ --.

Using essentially the conditions described above for the preparation ofthe diketone compounds of Formula VI, the 9-hydroxyenamine intermediatesof Formula XXV are converted to the diketone compounds of Formula XIV.Note that in this instance the reaction is effective for simultaneoushydrolysis of the enamine structure and dehydration at the 9,11positions to introduce the 9,11 double bond. The compound of Formula XIVis then converted to the compound of Formula XXXI, and thence to thecompound of Formula XIII, using the same steps that are described abovein scheme 3.

In a particularly preferred embodiment, the overall process of Scheme 5proceeds as follows: ##STR113##

Scheme 6

Scheme 6 provides an advantageous method for the preparation ofepoxymexrenone and other compounds corresponding to Formula I, startingwith 11α-hydroxylation of androstendione or other compound of FormulaXXXV ##STR114## wherein --A--A--, R³, and --B--B-- are as defined inFormula XIII, producing an intermediate corresponding to the FormulaXXXVI ##STR115## where --A--A--, R³, and --B--B-- are as defined inFormula XIII. Except for the selection of substrate, the process forconducting the 11α-hydroxylation is essentially as described hereinabovefor Scheme 1. The following microorganisms are capable of carrying outthe 11α-hydroxylation of androstendione or other compound of FormulaXXXV:

Aspergillus ochraceus NRRL 405 (ATCC 18500);

Aspergillus niger ATCC 11394;

Aspergillus nidulans ATCC 11267;

Rhizopus oryzae ATCC 11145;

Rhizopus stolonifer ATCC 6227b;

Trichothecium roseum ATCC 12519 and ATCC 8685.

11α-Hydroxyandrost-4-ene-3,17-dione, or other compound of Formula XXXVI,is next converted to 11α-hydroxy-3,4-enol ether of Formula (101):##STR116## where --A--A--, R³, and --B--B--, are as defined in FormulaXIII and R¹¹ is methyl or other lower alkyl (C₁ to C₄), by reaction withan etherifying reagent such as trialkyl orthoformate in the presence ofan acid catalyst. To carry out this conversion, the 11α-hydroxysubstrate is acidified by mixing with an acid such as, e.g., benzenesulfonic acid hydrate or toluene sulfonic acid hydrate and dissolved ina lower alcohol solvent, preferably ethanol. A trialkyl orthoformate,preferably triethyl orthoformate is introduced gradually over a periodof 5 to 40 minutes while maintaining the mixture in the cold, preferablyat about 0° C. to about 15° C. The mixture is then warmed and thereaction carried out at a temperature of between 20° C. and about 60° C.Preferably the reaction is carried out at 30° to 50° C. for 1 to 3hours, then heated to reflux for an additional period, typically 2 to 6hours, to complete the reaction. Reaction mixture is cooled, preferablyto 0° to 15°, preferably about 5° C., and the solvent removed undervacuum.

Using the same reaction scheme as described in Scheme 3, above, for theconversion of the compound of Formula XX to the compound of FormulaXVII, a 17-spirolactone moiety of Formula XXXIII is introduced into thecompound of Formula 101. For example, the Formula 101 substrate may bereacted with a sulfonium ylide in the presence of a base such as analkali metal hydroxide in a suitable solvent such as DMSO, to produce anintermediate corresponding to Formula 102: ##STR117## where --A--A--,R³, R¹¹, and --B--B-- are as defined in Formula 101. The intermediate ofFormula 102 is then reacted with a malonic acid diester in the presenceof an alkali metal alkoxide to form the five membered spirolactone ringand produce the intermediate of Formula 103 ##STR118## where --A--A--,R³, R¹¹, R¹², and --B--B-- are as defined in Formula XIII. Finally, thecompound of Formula 103 in a suitable solvent, such asdimethylformamide, is subjected to heat in the presence of an alkalimetal halide, splitting off the alkoxycarbonyl moiety and producing theintermediate of Formula 104: ##STR119## where again --A--A--, R³, R¹¹and --B--B-- are as defined in Formula XIII.

Next the 3,4-enol ether compound 104 is converted to the compound ofFormula XXIII, i.e., the compound of Formula VIII in which R⁸ and R⁹together form the moiety of Formula XXXIII, This oxidation step iscarried out in essentially the same manner as the oxidation step forconversion of the compound of Formula XXIV to the intermediate ofFormula XXIII in the synthesis of Scheme 4. Direct oxidation can beeffected using a reagent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) ortetrachlorobenzoquinone (chloranil), or preferably a two stage oxidationis effected by first brominating, e.g., with an N-halo brominating agentsuch as N-bromosuccinamide or 1,3-dibromo-5,5-dimethyl hydantoin (DBDMH)and then dehydrobrominating with a base, for example with DABCO in thepresence of LiBr and heat. Where NBS is used for bromination, an acidmust also be employed to convert 3-enol ether to the enone. DBDMH, anionic rather than free radical bromination reagent, is effective byitself for bromination and conversion of the enol ether to the enone.

The compound of Formula VIII is then converted to epoxymexrenone orother compound of Formula I by the steps described hereinabove forScheme 1.

Each of the intermediates of Formulae 101, 102, 103, and 104 is a novelcompound having substantial value as an intermediate for epoxymexrenoneor other compounds of Formulae IA and I. In each of the compounds ofFormulae 101, 102, 103, and 104 --A--A-- and --B--B-- are preferably--CH₂ --CH₂ -- and R³ is hydrogen, lower alkyl or lower alkoxy. Mostpreferably, the compound of Formula 101 is3-ethoxy-11α-hydroxyandrost-3,5-dien-17-one, the compound of Formula 102is 3-ethoxyspiro[androst-3,5-diene-17β,2'-oxiran]-11α-ol, the compoundof Formula 103 is ethyl hydrogen3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dicarboxylate,gamma-lactone, and the compound of Formula 104 is3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid,gamma-lactone.

In a particularly preferred embodiment, the overall process of Scheme 6proceeds as follows: ##STR120##

Scheme 7

Scheme 7 provides for the synthesis of epoxymexrenone and othercompounds of Formula I using a starting substrate comprisingβ-sitosterol, cholesterol, stigmasterol or other compound of FormulaXXXVII ##STR121## where --A--A--, R³, and --B--B-- are as defined inFormula XIII, D--D is --CH₂ --CH₂ -- or --CH═CH--, and each of R¹³, R¹⁴,R¹⁵ and R¹⁶ is independently selected from among hydrogen or C₁, to C₄alkyl.

In the first step of the synthesis 11α-hydroxyandrostendione or othercompound of Formula XXXV is prepared by bioconversion of the compound ofFormula XXXVII. The bioconversion process is carried out substantiallyin accordance with the method described hereinabove for the11α-hydroxylation of canrenone (or other substrate of Formula XIII).

In the synthesis 11α-hydroxyandrostendione, 4-androstene-3,17-dione isinitially prepared by bioconversion of the compound of Formula XXXVII.This initial bioconversion may be carried out in the manner described inU.S. Pat. No. 3,759,791, which is expressly incorporated herein byreference. Thereafter, 4-androstene-3,17-dione is converted to11α-hydroxyandrostenedione substantially in accordance with the methoddescribed hereinabove for the 11α-hydroxylation of canrenone (or othersubstrate of Formula XIII).

The remainder of the synthesis of Scheme 7 is identical to Scheme 6. Ina particularly preferred embodiment, the overall process of Scheme 7proceeds as follows: ##STR122##

The methods, processes and compositions of the invention, and theconditions and reagents used therein, are further described in thefollowing examples.

EXAMPLE 1

Slants were prepared with a growth medium as set forth in Table 1

                  TABLE 1                                                         ______________________________________                                        Y P D A                                                                         (medium for slants and plates)                                              ______________________________________                                        yeast extract          20     g                                                 peptone 20 g                                                                  glucose 20 g                                                                  agar 20 g                                                                     distilled water, q.s. to 1000 ml                                              pH as is 6.7                                                                  adjust at pH 5 with H.sub.3 PO.sub.4 10%                                      w/v                                                                           Distribute                                                                    for slants:                                                                   7.5 ml in 180 × 18 mm tubes                                             for plates (10 cm of φ)                                                   25 ml in 200 × 20 mm tubes                                              sterilize at 120° C. for 20                                            minutes                                                                       pH after sterilization: 5                                                   ______________________________________                                    

To produce first generation cultures, a colony of Aspergillus ochraceuswas suspended in distilled water (2 ml) in a test tube; and 0.15 mlaliquots of this suspension applied to each of the slants that had beenprepared as described above. The slants were incubated for seven days at25° C., after which the appearance of the surface culture was that of awhite cottony mycelium. The reverse was pigmented in orange in the lowerpart, in yellow-orange in the upper part.

The first generation slant cultures were suspended in a sterile solution(4 ml) containing Tween 80 nonionic surfactant (3% by weight), and 0.15ml aliquots of this suspension were used to inoculate second generationslants that had been prepared with the growth medium set forth in Table2

                  TABLE 2                                                         ______________________________________                                        (for second generation and routine slants)                                    ______________________________________                                        malt extract           20     g                                                 peptone 1 g                                                                   glucose 20 g                                                                  agar 20 g                                                                     distilled water q.s. to 1000 ml                                               pH as is 5.3                                                                  distribute in tubes (180 ×                                              18 mm) ml 7.5                                                                 sterilize at 120° C. for 20                                            minutes                                                                     ______________________________________                                    

The second generation slants were incubated for 10 days at 25° C.,producing a heavy mass of golden-colored spores; reverse pigmented inbrown orange.

A protective medium was prepared having the composition set forth inTable 3.

                  TABLE 3                                                         ______________________________________                                        PROTECTIVE MEDIUM                                                             ______________________________________                                        Skim milk              10     g                                                 distilled water 100 ml                                                        In a 250 ml flask                                                             containing 100 ml                                                             of distilled water                                                            at 50° C., add skim                                                    milk. Sterilize at                                                            120° C. for 15                                                         minutes. Cool at                                                              33° C. and use before                                                  the day is over                                                             ______________________________________                                    

Cultures from five of the second generation slants were suspended in theprotective solution (15 ml) in a 100 ml flask. The suspension wasdistributed in aliquots (0.5 ml each) among 100×10 mm tubes forlyophilization. These were pre-frozen at -70° to -80° C. in anacetone/dry ice bath for 20 minutes, then transferred immediately to adrying room pre-cooled to -40° to -50° C. The pre-frozen aliquots werelyophilized at a residual pressure of 50μ Hg and ≦-30° C. At the end ofthe lyophilization, two to three granules of sterile silica gel wereadded to each tube with moisture indicator and flame seal.

To obtain mother culture slants suitable for industrial scalefermentation, a single aliquot of lyophilized culture, which had beenprepared in the manner described above, was suspended in distilled water(1 ml) and 0.15 ml aliquots of the suspension were used to inoculateslants that had been provided with a growth medium having thecomposition set forth in Table 2. The mother slants were incubated forseven days at 25° C. At the end of incubation, the culture developed onthe slants was preserved at 4° C.

To prepare a routine slant culture, the culture from a mother slant wassuspended in a sterile solution (4 ml) containing Tween 80 (3% byweight) and the resulting suspension distributed in 0.15 ml aliquotsamong slants which had been coated with the growth medium described inTable 2. The routine slant cultures may be used to inoculate the primaryseed flasks for laboratory or industrial fermentations.

To prepare a primary seed flask culture, the culture from a routineslant, which had been prepared as described above, was removed andsuspended in a solution (10 ml) containing Tween 80 (3% by weight). A0.1 aliquot of the resulting suspension was introduced into a 500 mlbaffled flask containing a growth medium having the composition setforth in Table 4.

                  TABLE 4                                                         ______________________________________                                        (for primary and transformation flask                                           culture and round bottomed flask)                                           ______________________________________                                        glucose            20 g                                                         peptone 20 g                                                                  yeast autolysate 20 g                                                         distilled water q.s to                                                        pH as is 5.2                                                                  adjust at pH 5.8 with NaOH 20%                                                distribute in 500 ml baffled                                                  flask 100 ml                                                                  distribute in 2000 ml round                                                   bottomed flasks (3 baffles)                                                   500 ml                                                                        sterilize 120° C. × 20 min.                                      pH after sterilization                                                        about 5.7                                                                   ______________________________________                                    

The seed flask was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 24 hours at 28° C., thereby producing a culture in theform of pellet-like mycelia having diameters of 3 to 4 mm. Onmicroscopic observation, the seed culture was found to be a pureculture, with synnematic growth, with big hyphae and well twisted. ThepH of the suspension was 5.4 to 5.6. PMV was 5 to 8% as determined bycentrifugation (3000 rpm×5 min.).

A transformation flask culture was prepared by inoculating a growthmedium (100 ml) having the composition set forth Table 4 in a second 500ml shaker flask with biomass (1 ml) from the seed culture flask. Theresulting mixture was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 18 hours at 28° C. The culture was examined and foundto comprise pellet like mycelia with a 3-4 mm diameter. On microscopicexamination, the culture was determined to be a pure culture, withsynnematic and filamentous growth in which the apical cells were full ofcytoplasm and the olden cells were little vacuolated. The pH of theculture suspension was 5 to 5.2 and the PMV was determined bycentrifugation to be between 10% and 15%. Accordingly, the culture wasdeemed suitable for transformation of canrenone to 11α-hydroxycanrenone.

Canrenone (1 g) was micronized to about 5μ and suspended in sterilewater (20 ml). To this suspension were added: a 40% (w/v) sterileglucose solution; a 16% (w/v) sterile solution of autolyzed yeast; and asterile antibiotic solution; all in the proportions indicated for 0hours reaction time in Table 5. The antibiotic solution had beenprepared by dissolving kanamicyn sulfate (40 mg), tetracycline HCl (40mg) and cefalexin (200 mg) in water (100 ml). The steroid suspension,glucose solution, and autolyzed yeast solution were added gradually tothe culture contained in the shaker flask.

                  TABLE 5                                                         ______________________________________                                        Indicative Additions of Steroid and Solutions                                   (additives and antibiotics) in the Course                                     of Bioconversion of Canrenone in Shake Flask                                  Reaction Steroid Suspension                                                                          glucose                                                                              yeast  antibiotic                             time            approx.  solution                                                                             autolised                                                                            solution                                 hours ml mg. ml sol. ml. ml                                                 ______________________________________                                         0     1         50      1      0.5    1                                         8 2 100 2 1                                                                  24 2 100 1 0.5 1                                                              32 5 250 2 1                                                                  48 2 100 1 0.5 1                                                              56 5 250 2 1                                                                  72 3 150 1 0.5 1                                                              90                                                                          ______________________________________                                    

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Additional canrenone substrate andnutrients were added to the fermentation reaction mixture during thereaction at rates controlled to maintain the glucose content in therange of about 0.1% by weight. The addition schedule for steroidsuspension, glucose solution, autolyzed yeast solution and antibioticsolution is set forth in Table 5. The transformation reaction continuedfor 96 hours at 25° C. on a rotary shaker (200 rpm and 5 cmdisplacement). The pH ranged between 4.5 and 6 during the fermentation.Whenever the PMV rose to or above 60%, a 10 ml portion of broth culturewas withdrawn and replaced with 10 ml distilled water. The disappearanceof canrenone and appearance of 11α-hydroxycanrenone were monitoredduring the reaction by sampling the broth at intervals of 4, 7, 23, 31,47, 55, 71, 80, and 96 hours after the start of the fermentation cycle,and analyzing the sample by TLC. The progress of the reaction asdetermined from these samples is set forth in Table 6

                  TABLE 6                                                         ______________________________________                                        Time Course of Bioconversion                                                    of Canrenone in Shake Flask                                                           Transformation Ratio                                                Time      Canrenone Rf.                                                                            11 αhydroxy Canrenone                                hours    RF. = 0.81    RF. = 0.29                                           ______________________________________                                        0         100        0.0                                                        4        50                 50                                                7        20                 80                                                23       20                 80                                                31       30                 70                                                47       20                 80                                                55       30                 70                                                71       25                 75                                                80       15                 85                                                96       ˜10          ˜90                                       ______________________________________                                    

EXAMPLE 2

A primary seed flask culture was prepared in the manner described inExample 1. A nutrient mixture was prepared having the composition setforth in Table 7

                  TABLE 7                                                         ______________________________________                                        For Transformation Culture                                                      in 10 1 glass fermenter                                                                         quantity                                                                              g/l                                               ______________________________________                                        glucose             80    g     20                                              peptone 80 g 20                                                               yeast autolised 80 g 20                                                       antifoam SAG 471 0.5 g                                                        deionized water q.s. to 4 l                                                   sterilize the empty                                                           fermenter for 30                                                              minutes at 130° C.                                                     load it with 3 l of                                                           deionized water,                                                              heat at 40° C.                                                         add while stirring                                                            the components of                                                             the medium                                                                    stir for 15 minutes,                                                          bring to volume of                                                            3.9 l                                                                         pH as is 5.1                                                                  adjust of 5.8 with                                                            NaOH 20% w/v                                                                  sterilize at 120° C. ×                                           20 minutes                                                                    pH after                                                                      sterilization 5.5-                                                            5.7                                                                         ______________________________________                                    

An initial charge of this nutrient mixture (4 L) was introduced into atransformation fermenter of 10 L geometric volume. The fermenter was ofcylindrical configuration with a height to diameter ratio of 2.58. Itwas provided with a 400 rpm turbine agitator having two No. 2 diskwheels with 6 blades each. The external diameter of the impellers was 80mm, each of the blades was 25 mm in radial dimension and 30 mm high, theupper wheel was positioned 280 mm below the top of the vessel, the lowerwheel was 365 mm below the top, and baffles for the vessel were 210 mmhigh and extended radially inwardly 25 mm from the interior verticalwall of the vessel.

Seed culture (40 ml) was mixed with the nutrient charge in thefermenter, and a transformation culture established by incubation for 22hours at 28° C., and an aeration rate of 0.5 1/1-min. at a pressure of0.5 kg/cm². At 22 hours, the PMV of the culture was 20-25% and the pH 5to 5.2.

A suspension was prepared comprising canrenone (80 g) in sterile water(400 ml), and a 10 ml portion added to the mixture in the transformationfermenter. At the same time a 40% (w/v) sterile glucose solution, a 16%(w/v) sterile solution of autolyzed yeast, and a sterile antibioticsolution were added in the proportions indicated in Table 8 at 0 hoursreaction time. The antibiotic solution was prepared in the mannerdescribed in Example 1.

                  TABLE 8                                                         ______________________________________                                        Indicative Additions of Steroid and Solutions                                   (additives and antibiotics) in the Course                                     of Bioconversion of Canrenone in                                              10 l Glass Fermenter                                                                   Steroid              yeast  anti-                                    Reaction Suspension glucose autolised biotic                                time            approx  solution                                                                              solution                                                                             solution                                 hours ml gr ml ml ml                                                        ______________________________________                                         0     10       4       25      12.5   40                                        4   25 12.5                                                                   8 10   4 25 12.5                                                             12   25 12.5                                                                  16 10   4 25 12.5                                                             20   25 12.5                                                                  24 10   4 25 12.5 40                                                          28 10   4 25 12.5                                                             32 12.5 5 25 12.5                                                             36 12.5 5 25 12.5                                                             40 12.5 5 25 12.5                                                             44 12.5 5 25 12.5                                                             48 12.5 5 25 12.5 40                                                          52 12.5 5 25 12.5                                                             56 12.5 5 25 12.5                                                             60 12.5 5 25 12.5                                                             64 12.5 5 25 12.5                                                             68 12.5 5 25 12.5                                                             72 12.5 5 25 12.5 40                                                          76 12.5 5 25 12.5                                                             80                                                                            84                                                                            88                                                                          ______________________________________                                    

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Based on TLC analysis of reactionbroth samples as described hereinbelow, additional canrenone was addedto the reaction mixture as canrenone substrate was consumed. Glucoselevels were also monitored and, whenever glucose concentration droppedto about 0.05% by weight or below, supplemental glucose solution wasadded to bring the concentration up to about 0.25% by weight. Nutrientsand antibiotics were also added at discrete times during the reactioncycle. The addition schedule for steroid suspension, glucose solution,autolyzed yeast solution and antibiotic solution is set forth in Table8. The transformation reaction continued for 90 hours at an aerationrate of 0.5 vol. air per vol. liquid per minute (vvm) at a positive headpressure of 0.3 kg/cm². The temperature was maintained at 28° C. untilPVM reached 45%, then decreased to 26° C. and maintained at thattemperature as PVM grew from 45% to 60%, and thereafter controlled at24° C. The initial agitation rate was 400 rpm, increasing to 700 rpmafter 40 hours. The pH was maintained at between 4.7 and 5.3 byadditions of 2M orthophosphoric acid or 2M NaOH, as indicated. Foamingwas controlled by adding a few drops of Antifoam SAG 471 as foamdeveloped. The disappearance of canrenone and appearance of11α-hydroxycanrenone were monitored at 4 hour intervals during thereaction by TLC analysis of broth samples. When most of the canrenonehad disappeared from the broth, additional increments were added.

After all canrenone additions had been made, the reaction was terminatedwhen TLC analysis showed that the concentration of canrenone substraterelative to 11α-hydroxycanrenone product had dropped to about 5%.

At the conclusion of the reaction cycle, the fermentation broth wasfiltered through cheese cloth for separation of the mycelium from theliquid broth. The mycelia fraction was resuspended in ethyl acetateusing about 65 volumes (5.2 liters) per gram canrenone charged over thecourse of the reaction. The suspension of mycelia in ethyl acetate wasrefluxed for one hour under agitation, cooled to about 20° C., andfiltered on a Buchner. The mycelia cake was washed sequentially withethyl acetate (5 vol. per g canrenone charge; 0.4 L) and deionized water(500 ml) to displace the ethyl acetate extract from the cake. The filtercake was discarded. The rich extract, solvent washing and water washingwere collected in a separator, then allowed to stand for 2 hours forphase separation.

The aqueous phase was then discarded and the organic phase concentratedunder vacuum to a residual volume of 350 ml. The still bottoms werecooled to 15° C. and kept under agitation for about one hour. Theresulting suspension was filtered to remove the crystalline product, andthe filter cake was washed with ethyl acetate (40 ml) After drying, theyield of 11α-hydroxycanrenone was determined to be 60 g.

EXAMPLE 3

A spore suspension was prepared from a routine slant in the mannerdescribed in Example 1. In a 2000 ml baffled round bottomed flask (3baffles, each 50 mm×30 mm), an aliquot (0.5 ml) of the spore suspensionwas introduced into a nutrient solution (500 ml) having the compositionset forth in Table 4. The resulting mixture was incubated in the flaskfor 24 hours at 25° C. on an alternating shaker (120 strokes per min.;displacement 5 cm), thereby producing a culture which, on microscopicexamination, was observed to appear as a pure culture with hyphae welltwisted. The pH of the culture was between about 5.3 and 5.5, and thePMV (as determined by centrifugation at 3000 rpm for 5 min.) was 8 to10%.

Using the culture thus prepared, a seed culture was prepared in astainless steel fermenter of vertical cylindrical configuration, havinga geometric volume of 160 L and an aspect ratio of 2.31 (height=985 mm;diameter=425 mm). The fermenter was provided with a disk turbine typeagitator having two wheels, each wheel having six blades with anexternal diameter of 240 mm, each blade having a radial dimension of 80mm and a height of 50 mm. The upper wheel was positioned at a depth of780 mm from the top of the fermenter, and the second at a depth of 995mm. Vertical baffles having a height of 890 mm extended radiallyinwardly 40 mm from the interior vertical wall of the fermenter. Theagitator was operated at 170 rpm. A nutrient mixture (100 L) having thecomposition set forth in Table 9 was introduced into the fermenter,followed by a portion of preinoculum (1 L) prepared as described aboveand having a pH of 5.7.

                  TABLE 9                                                         ______________________________________                                        For Vegetative Culture in 160 L                                                 Fermenter About 8 L are needed                                                to Seed Productive fermenter                                                                   Quantity g/L                                               ______________________________________                                        glucose            2      kg    20                                              peptone 2 kg 20                                                               yeast autolysed 2 kg 20                                                       antifoam SAG 471 0.010 Kg traces                                              deionized water q.s. to 100 L                                                 sterilize the empty                                                           fermenter for 1 hour                                                          at 130° C.                                                             load it with 6 L of                                                           deionized water;                                                              heat at 40° C.                                                         add while stirring                                                            the components of                                                             the medium                                                                    stir for 15 minutes,                                                          bring to volume of                                                            95 L                                                                          sterilization at                                                              121° C. for 30 minutes                                                 post sterilization                                                            pH is 5.7                                                                     add sterile                                                                   deionized water                                                               to 100 L                                                                    ______________________________________                                    

The inoculated mixture was incubated for 22 hours at an aeration rate of0.5 L/L-min. at a head pressure of 0.5 kg/cm². The temperature wascontrolled at 28° C. until PMV reached 25%, and then lowered to 25° C.The pH was controlled in the range of 5.1 to 5.3. Growth of myceliumvolume is shown in Table 10, along with pH and dissolved oxygen profilesof the seed culture reaction.

                  TABLE 10                                                        ______________________________________                                        Time Course for Mycelial Growth in                                              Seed Culture Fermentation                                                                            packed mycelium                                        Fermentation  volume (pmv) % dissolved                                        period h pH (3000 rpms 5 min) oxygen %                                      ______________________________________                                         0        5.7 ± 0.1            100                                            4 5.7 ± 0.1  100                                                           8 5.7 ± 0.1 12 ± 3 85 ± 5                                           12 5.7 ± 0.1 15 ± 3 72 ± 5                                           16 5.5 ± 0.1 25 ± 5 40 ± 5                                           20 5.4 ± 0.1 30 ± 5 35 ± 5                                           22 5.3 ± 0.1 33 ± 5 30 ± 5                                           24 5.2 ± 0.1 35 ± 5 25 ± 5                                         ______________________________________                                    

Using the seed culture thus produced, a transformation fermentation runwas carried out in a vertical cylindrical stainless steel fermenterhaving a diameter of 1.02 m, a height of 1.5 m and a geometric volume of1.4 m³. The fermenter was provided with a turbine agitator having twoimpellers, one positioned 867 cm below the top of the reactor and theother positioned 1435 cm from the top. Each wheel was provided with sixblades, each 95 cm in radial dimension and 75 cm high. Vertical baffles1440 cm high extended radially inwardly 100 cm from the interiorvertical wall of the reactor. A nutrient mixture was prepared having thecomposition set forth in Table 11

                  TABLE 11                                                        ______________________________________                                        For Bioconversion Culture                                                       in 1000 L Fermenter                                                                            Quantity g/L                                               ______________________________________                                        glucose            16     kg    23                                              peptone 16 kg 23                                                              yeast autolysed 16 kg 23                                                      antifoam SAG 471 0.080 Kg traces                                              deionized water q.s. to 700 L                                                 sterilize the empty                                                           fermenter for 1 hour                                                          at 130° C.                                                             load it with 600 L                                                            of deionized water;                                                           heat at 40° C.                                                         add while stirring                                                            the components of                                                             the medium                                                                    stir for 15 minutes,                                                          bring to volume of                                                            650 L                                                                         sterilization at                                                              121° C. for 30 minutes                                                 post sterilization                                                            pH is 5.7                                                                     add sterile                                                                   deionized water                                                               to 700 L                                                                    ______________________________________                                    

An initial charge (700 L) of this nutrient mixture (pH=5.7) wasintroduced into the fermenter, followed by the seed inoculum of thisexample (7 L) prepared as described above.

The nutrient mixture containing inoculum was incubated for 24 hours atan aeration rate of 0.5 L/L-min at a head pressure of 0.5 kg/cm². Thetemperature was controlled at 28° C., and the agitation rate was 110rpm. Growth of mycelium volume is shown in Table 12, along with pH anddissolved oxygen profiles of the seed culture reaction.

                  TABLE 12                                                        ______________________________________                                        Time Course for Mycelial Growth in                                              Fermenter of the Transformation Culture                                                              packed mycelium                                        Fermentation  volume (pmv) % dissolved                                        period h pH (3000 rpm × 5 min) oxygen %                               ______________________________________                                         0        5.6 ± 0.2            100                                            4 5.5 ± 0.2  100                                                           8 5.5 ± 0.2 12 ± 3 95 ± 5                                           12  15 ± 3 90 ± 5                                                       16 5.4 ± 0.1 20 ± 5 75 ± 5                                           20 5.3 ± 0.1 25 ± 5 60 ± 5                                           22 5.2 ± 0.1 30 ± 5 40 ± 5                                         ______________________________________                                    

At the conclusion of the incubation, pelleting of the mycelium wasobserved, but the pellets were generally small and relatively looselypacked. Diffuse mycelium was suspended in the broth. Final pH was 5.1 to5.3.

To the transformation culture thus produced was added a suspension ofcanrenone (1.250 kg; micronized to 5μ) in sterile water (5 L). Sterileadditive solution and antibiotic solution were added in the proportionsindicated at reaction time 0 in Table 14. The composition of theadditive solution is set forth in Table 13.

                  TABLE 13                                                        ______________________________________                                        ADDITIVE SOLUTION                                                               (for transformative culture)                                                                       quantity                                               ______________________________________                                        dextrose               40     Kg                                                yeast autolysate 8 Kg                                                         antifoam SAG 471 0.010 Kg                                                     deionized water q.s. to 100 l                                                 sterilize a 150 l empty                                                       fermenter for 1 hour at                                                       130° C.                                                                load it with 70 l of                                                          deionized water; heat                                                         at 40° C.                                                              add while stirring the                                                        components of "additive                                                       solution"                                                                     stir for 30 minutes,                                                          bring to volume of 95 l                                                       pH as is 4.9                                                                  sterilize at 120° C. ×                                           20 minutes                                                                    pH after sterilization                                                        about 5                                                                     ______________________________________                                    

Bioconversion was carried out for about 96 hours with aeration at 0.5L/L-min. at a head pressure of 0.5 kg/cm² and a pH of ranging between4.7 and 5.3, adjusted as necessary by additions of 7.5 M NaOH or 4 M H₃PO₄. The agitation rate was initially 100 rpm, increased to 165 rpm at40 hours and 250 rpm at 64 hours. The initial temperature was 28° C.,lowered to 26° C. when PMV reached 45%, and lowered to 24° C. when PMVrose to 60%. SAG 471 in fine drops was added as necessary to controlfoaming. Glucose levels in the fermentation were monitored at 4 hourintervals and, whenever the glucose concentration fell below 1 gpl, anincrement of sterile additive solution (10 L) was added to the batch.Disappearance of canrenone and appearance of 11α-hydroxycanrenone werealso monitored during the reaction by HPLC. When at least 90% of theinitial canrenone charge had been converted to 11α-hydroxycanrenone, anincrement of 1.250 kg canrenone was added. When 90% of the canrenone inthat increment was shown to have been converted, another 1.250 kgincrement was introduced. Using the same criterion further increments(1.250 kg apiece) were added until the total reactor charge (20 kg) hadbeen introduced. After the entire canrenone charge had been delivered tothe reactor, reaction was terminated when the concentration of unreactedcanrenone was 5% relative to the amount of 11-hydroxycanrenone produced.The schedule for addition of canrenone, sterile additive solution, andantibiotic solution is as shown in Table 14.

                  TABLE 14                                                        ______________________________________                                        Additions of the Steroid and Solutions                                          (additives and antibiotics)                                                   in the Course of Bioconversion of Canrenone                                   in Fermenter                                                                           CANRENONE     Sterile anti-                                          Reaction in suspension additive biotic volume                               time            Progress-                                                                              solution                                                                              solution                                                                            liters                                   hours Kg ive Kg liters liters about                                         ______________________________________                                        0      1.250    1.25     10      8     700                                      4   10                                                                        8 1.250 2.5 10                                                                12   10                                                                       16 1.250  10                                                                  20   10                                                                       24 1.250 5 10 8 800                                                           28 1.250  10                                                                  32 1.250  10                                                                  36 1.250  10                                                                  40 1.250  10                                                                  44 1.250  10                                                                  48 1.250 12.5 10 8 900                                                        52 1.250  10                                                                  56 1.250  10                                                                  60 1.250  10                                                                  64 1.250  10                                                                  68 1.250  10                                                                  72 1.250 20 10 8 1050                                                         76    0                                                                       80                                                                            84                                                                            88                                                                            92                                                                            Total                                                                       ______________________________________                                    

When bioconversion was complete, the mycelia were separated from thebroth by centrifugation in a basket centrifuge. The filtrate wasdetermined by HPLC to contain only 2% of the total quantity of11α-hydroxycanrenone in the harvest broth, and was therefore eliminated.The mycelia were suspended in ethyl acetate (1000 L) in an extractiontank of 2 m³ capacity. This suspension was heated for one hour underagitation and ethyl acetate reflux conditions, then cooled andcentrifuged in a basket centrifuge. The mycelia cake was washed withethyl acetate (200 L) and thereafter discharged. The steroid richsolvent extract was allowed to stand for one hour for separation of thewater phase. The water phase was extracted with a further amount ofethyl acetate solvent (200 L) and then discarded. The combined solventphases were clarified by centrifugation and placed in a concentrator(500 L geometric volume) and concentrated under vacuum to a residualvolume of 100 L. In carrying out the evaporation, the initial charge tothe concentrator of combined extract and wash solutions was 100 L, andthis volume was kept constant by continual or periodic additions ofcombined solution as solvent was taken off. After the evaporation stepwas complete, the still bottoms were cooled to 20° C. and stirred fortwo hours, then filtered on a Buchner filter. The concentrator pot waswashed with ethyl acetate (20 L) and this wash solution was then used towash the cake on the filter. The product was dried under vacuum for 16hours at 50° C. Yield of 11α-hydroxycanrenone was 14 kg.

EXAMPLE 4

Lyophilized spores of Aspergillus ochraceus NRRL 405 were suspended in acorn steep liquor growth medium (2 ml) having the composition set forthin Table 15:

                  TABLE 15                                                        ______________________________________                                        Corn Steep Liquor Medium                                                        (Growth Medium for Primary Seed Cultivation)                                ______________________________________                                        Corn steep liquor      30 g                                                     Yeast extract 15 g                                                            Ammonium phosphate  3 g                                                       Monobasic                                                                     Glucose (charge after sterilization) 30 g                                     distilled water, q.s. to 1000 ml                                              pH as is: 4.6, adjust to pH 6.5 with                                          20% NaOH, distribute 50 ml to 250 ml                                          Erlenmeyer flask sterilize 121° C. for                                 20 minutes.                                                                 ______________________________________                                    

The resulting suspension was used in an inoculum for the propagation ofspores on agar plates. Ten agar plates were prepared, each bearing asolid glucose/yeast extract/phosphate/agar growth medium having thecomposition set forth in Table 16:

                  TABLE 16                                                        ______________________________________                                        GYPA                                                                            (Glucose/Yeast Extract/Phosphate                                              Agar for Plates)                                                            ______________________________________                                        Glucose (charge after sterilization)                                                                   10    g                                                Yeast extract 2.5 g                                                           K.sub.2 HPO.sub.4 3 g                                                         Agar 20 g                                                                     distilled water, q.s. to 1000 ml                                              adjust pH to 6.5                                                              sterilize 121° C. for 30 minutes                                     ______________________________________                                    

A 0.2 ml aliquot of the suspension was transferred onto the surface ofeach plate. The plates were incubated at 25° C. for ten days, afterwhich the spores from all the plates were harvested into a sterilecryogenic protective medium having the composition set forth in Table17:

                  TABLE 17                                                        ______________________________________                                        GYP/Glycerol                                                                    (Glucose/Yeast Extract/                                                       Phosphate/Glycerol                                                            medium for stock vials)                                                     ______________________________________                                        Glucose (charge after sterilization)                                                                   10    g                                                Yeast extract 2.5 g                                                           K.sub.2 HPO.sub.4 3 g                                                         Glycerol 20 g                                                                 Distiiled water, q.s. to 1000 mL                                              Sterilize at 121° C. for 30 minutes                                  ______________________________________                                    

The resulting suspension was divided among twenty vials, with one mlbeing transferred to each vial. These vials constitute a master cellbank that can be drawn on to produce working cell banks for use ingeneration of inoculum for bioconversion of canrenone to11α-hydroxycanrenone. The vials comprising the master cell bank werestored in the vapor phase of a liquid nitrogen freezer at -130° C.

To begin preparation of a working cell bank, the spores from a singlemaster cell bank vial were resuspended in a sterile growth medium (1 ml)having the composition set forth in Table 15. This suspension wasdivided into ten 0.2 ml aliquots and each aliquot used to inoculate anagar plate bearing a solid growth medium having the composition setforth in Table 16. These plates were incubated for ten days at 25° C. Bythe third day of incubation, the underside of the growth medium wasbrown-orange. At the end of the incubation there was heavy production ofgolden colored spores. The spores from each plate were harvested by theprocedure described hereinabove for the preparation of the master cellbank. A total of one hundred vials was prepared, each containing 1 ml ofsuspension. These vials constituted the working cell bank. The workingcell bank vials were also preserved by storage in the vapor phase of aliquid nitrogen freezer at -130° C.

Growth medium (50 ml) having the composition set forth in Table 15 wascharged to a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) of workingcell suspension was introduced into the flask and mixed with the growthmedium. The inoculated mixture was incubated for 24 hours at 25° C. toproduce a primary seed culture having a percent packed mycelial volumeof approximately 45% Upon visual inspection the culture was found tocomprise pellet-like mycelia of 1 to 2 mm diameter; and upon microscopicobservation it appeared as a pure culture.

Cultivation of a secondary seed culture was initiated by introducing agrowth medium having the composition set forth in Table 15 into a 2.8 LFernbach flask, and inoculating the medium with a portion (10 ml) of theprimary seed culture of this example, the preparation of which was asdescribed above. The inoculated mixture was incubated at 25° C. for 24hours on a rotating shaker (200 rpm, 5 cm displacement). At the end ofthe incubation, the culture exhibited the same properties as describedabove for the primary seed culture, and was suitable for use in atransformation fermentation in which canrenone was bioconverted to11α-hydroxycanrenone.

Transformation was conducted in a Braun E Biostat fermenter configuredas follows:

Capacity: 15 liters with round bottom

Height: 53 cm

Diameter: 20 cm

H/D: 2.65

Impellers: 7.46 cm diameter, six paddles 2.2×1.4 cm each

Impeller spacing: 65.5, 14.5 and 25.5 cm from bottom of tank

Baffles: four 1.9×48 cm

Sparger: 10.1 cm diameter, 21 holes ˜1 mm diameter

Temperature control: provided by means of an external vessel jacket

Canrenone at a concentration of 20 g/L was suspended in deionized water(4 L) and a portion (2 L) of growth medium having the composition setforth in Table 18 was added while the mixture in the fermenter wasstirred at 300 rpm.

                  TABLE 18                                                        ______________________________________                                        (Growth medium for bioconversion                                                culture in 10 L fermenter)                                                                         Quantity   Amount/L                                    ______________________________________                                        glucose (charge after                                                                            160 g      20 g                                              sterilization)                                                                peptone 160 g 20 g                                                            yeast extract 160 g 20 g                                                      antifoam SAF471 4.0 ml 0.5 ml                                                 Canrenone 160 g 20 g                                                          deionized water q.s. to 7.5 L                                                 sterilize 121° C. for 30                                               minutes                                                                     ______________________________________                                    

The resulting suspension was stirred for 15 minutes, after which thevolume was brought up to 7.5 L with additional deionized water. At thispoint the pH of the suspension was adjusted from 5.2 to 6.5 by additionof 20% by weight NaOH solution, and the suspension was then sterilizedby heating at 121° C. for 30 minutes in the Braun E fermenter. The pHafter sterilization was 6.3±0.2, and the final volume was 7.0 L. Thesterilized suspension was inoculated with a portion (0.5 L) of thesecondary seed culture of this example that has been prepared asdescribed above, and the volume brought up to 8.0 L by addition of 50%sterile glucose solution. Fermentation was carried out at a temperatureof 28° C. until the PMV reached 50%, then lowered to 26° C., and furtherlowered to 24° C. when PMV exceeded 50% in order to maintain aconsistent PMV below about 60%. Air was introduced through the spargerat a rate of 0.5 vvm based on initial liquid volume and the pressure inthe fermenter was maintained at 700 millibar gauge. Agitation began at600 rpm and was increased stepwise to 1000 rpm as needed to keep thedissolved oxygen content above 30% by volume. Glucose concentration wasmonitored. After the initial high glucose concentration fell below 1%due to consumption by the fermentation reaction, supplemental glucosewas provided via a 50% by weight sterile glucose solution to maintainthe concentration in the 0.05% to 1% range throughout the remainder ofthe batch cycle. Prior to inoculation the pH was 6.3±0.2. After the pHdropped to about 5.3 during the initial fermentation period, it wasmaintained in the range of 5.5±0.2 for the remainder of the cycle byaddition of ammonium hydroxide. Foam was controlled by adding apolyethylene glycol antifoam agent sold under the trade designation SAG471 by OSI Specialties, Inc.

Growth of the culture took place primarily during the first 24 hours ofthe cycle, at which time the PMV was about 40%, the pH was about 5.6 andthe dissolved oxygen content was about 50% by volume. Canrenoneconversion began even as the culture was growing. Concentrations ofcanrenone and 11α-hydroxycanrenone were monitored during thebioconversion by analyzing daily samples. Samples were extracted withhot ethyl acetate and the resulting sample solution analyzed by TLC andHPLC. The bioconversion was deemed complete when the residual canrenoneconcentration was about 10% of the initial concentration. Theapproximate conversion time was 110 to 130 hours.

When bioconversion was complete, mycelial biomass was separated from thebroth by centrifugation. The supernatant was extracted with an equalvolume of ethyl acetate, and the aqueous layer discarded. The mycelialfraction was resuspended in ethyl acetate using approximately 65 volumesper g canrenone charged to the fermentation reactor. The mycelialsuspension was refluxed for one hour under agitation, cooled to about20° C., and filtered on a Buchner funnel. The mycelial filter cake waswashed twice with 5 volumes of ethyl acetate per g of canrenone chargedto the fermenter, and then washed with deionized water (1 L) to displacethe residual ethyl acetate. The aqueous extract, rich solvent, solventwashing and water washing were combined. The remaining exhaustedmycelial cake was either discarded or extracted again, depending onanalysis for residual steroids therein. The combined liquid phases wereallowed to settle for two hours. Thereafter, the aqueous phase wasseparated and discarded, and the organic phase concentrated under vacuumuntil the residual volume was approximately 500 ml. The still bottle wasthen cooled to about 15° C. with slow agitation for about one hour. Thecrystalline product was recovered by filtration, and washed with chilledethyl acetate (100 ml). Solvent was removed from the crystals byevaporation, and the crystalline product dried under vacuum at 50° C.

EXAMPLE 5

Lyophilized spores of Aspergillus ochraceus ATCC 18500 were suspended ina corn steep liquor growth medium (2 ml) as described in Example 4. Tenagar plates were prepared, also in the manner of Example 4. The plateswere incubated and harvested as described in Example 4 to provide amaster cell bank. The vials comprising the master cell bank were storedin the vapor phase of a liquid nitrogen freezer at -130° C.

From a vial of the master cell bank, a working cell bank was prepared asdescribed in Example 4, and stored in the nitrogen freezer at -130° C.

Growth medium (300 mL) having the composition set forth in Table 19 wascharged to a 2 L baffled flask. An aliquot (3 mL) of working cellsuspension was introduced into the flask. The inoculated mixture wasincubated for 20 to 24 hours at 28° C. on a rotating shaker (200 rpm, 5cm displacement) to produce a primary seed culture having a percentpacked mycelial volume of approximately 45%. Upon visual inspection theculture was found to comprise pell et like mycelia of 1 to 2 mmdiameter; and upon microscopic observation it appeared as a pureculture.

                  TABLE 19                                                        ______________________________________                                        Growth medium for primary and                                                   secondary seed cultivation                                                                          Amount/L                                              ______________________________________                                        glucose (charge after                                                                             20 g                                                        sterilization)                                                                peptone 20 g                                                                  Yeast extract 20 g                                                            distilled water q.s. to 1000 mL                                               sterilize 121° C. for 30 minutes                                     ______________________________________                                    

Cultivation of a secondary seed culture was initiated by introducing 8 Lgrowth medium having the composition set forth in Table 19 into a 14 Lglass fermenter. Inoculate the fermenter with 160 mL to 200 mL of theprimary seed culture of this example. The preparation of which was asdescribed above.

The inoculated mixture was cultivated at 28° C. for 18-20 hours, 200 rmpagitation, aeration rate was 0.5 vvm. At the end of the propagation, theculture exhibited the same properties as described above for the primaryseed.

Transformation was conducted in a 60 L fermenter, substantially in themanner described in Example 4, except that the growth medium had thecomposition set forth in Table 20, and the initial charge of secondaryseed culture was 350 mL to 700 mL. Agitation rate was initially 200 rpm,but increased to 500 rpm as necessary to maintain dissolved oxygen above10% by volume. The approximate bioconversion time for 20 g/L canrenonewas 80 to 160 hours.

                  TABLE 20                                                        ______________________________________                                        Growth Medium for Bioconversion                                                 Culture in 60 L Fermenter                                                                           Quantity   Amount/L                                   ______________________________________                                        glucose (charge after                                                                             17.5 g     0.5 g                                            sterilization)                                                                peptone 17.5 g 0.5 g                                                          yeast extract 17.5 g 0.5 g                                                    Canrenone (charge as a 700 g 20 g                                             20% slurry in sterile                                                         water)                                                                        deionized water, q.s. to 35 L                                                 sterilize 121° C. for 30 minutes                                     ______________________________________                                    

EXAMPLE 6

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. Using secondary seed culture produced in this manner, twobioconversion runs were made in accordance with a modified process ofthe type illustrated in FIG. 1, and two runs were made with the processillustrated in FIG. 2. The transformation growth medium, canrenoneaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Table 21. Run R2A used a canrenone addition schemebased on the same principle as Example 3, while run R2C modified theExample 3 scheme by making only two additions of canrenone, one at thebeginning of the batch, and one after 24 hours. In runs R2B and R2D, theentire canrenone charge was introduced at the beginning of the batch andthe process generally carried in the manner described in Example 4,except that the canrenone charge was sterilized in a separate vesselbefore it was charged to the fermenter and glucose was added as thebatch progressed. A Waring blender was used to reduce chunks produced onsterilization In runs R2A and R2B, canrenone was introduced into thebatch in methanol solution, in which respect these runs further differedfrom the runs of Examples 3 and 4, respectively.

                                      TABLE 21                                    __________________________________________________________________________    Descriptions of the Initial Canrenone Bioconversion Processes                 Run Number                                                                            R2A       R2B       R2C     R2D                                       __________________________________________________________________________    Medium (g/L)                                                                    Corn steep liq. 30 the same as run 30 the same as run                         Yeast extract 15 R2A 15 R2C                                                   NH.sub.4 H.sub.2 PO.sub.4 3  3                                                Glucose 15  30                                                                OSA 0.5 ml  0.5 ml                                                            pH adjusted to 6.0  adjusted to                                                with 2.5N NaOH  6.5 with                                                        2.5N NaOH                                                                  Canrenone 10 g/80 ml MEOH 80 g/640 ml MEOH Sterilized and Sterilized                                            and                                          added at 0, 18, added at 0 hr all blended; added blended; added                                                  24, 30, 36, 42, at once at: 0 hr:                                           25 g at: 0 hr: 200 g                         50, 56, 62 and  24 hr: 200 g                                                  68 hr.                                                                       Harvest time 143 hrs. 166 hrs. 125 hrs. 104 hrs.                              Bioconversion 45.9% 95.6% 98.1% 95.1%                                       __________________________________________________________________________

In runs R2A and R2B, the methanol concentration accumulated to about6.0% in the fermentation beer, which was found to be inhibitory to thegrowth of culture and bioconversion. However, based on the results ofthese runs, it was concluded that methanol or other water-misciblesolvent could serve effectively at lower concentrations to increase thecanrenone charge and provide canrenone as a fine particle precipitateproviding a large interfacial area for supply of canrenone to thesubject to the reaction.

Canrenone proved stable at sterilization temperature (121° C.) butaggregated into chunks. A Waring blender was employed to crush the lumpsinto fine particles, which were successfully converted to product.

EXAMPLE 7

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. The description and results of Example 7 are shown inTable 22. Using secondary seed culture produced in this manner, onebioconversion (R3C) was carried out substantially as described inExample 3, and three bioconversions were carried out in accordance withthe process generally described in Example 5. In the latter three runs(R3A, R3B and R3D), canrenone was sterilized in a portable tank,together with the growth medium except for glucose. Glucose wasaseptically fed from another tank. The sterilized canrenone suspensionwas introduced into the fermenter either before inoculation or duringthe early stage of bioconversion. In run R3B, supplemental sterilecanrenone and growth medium was introduced at 46.5. Lumps of canrenoneformed on sterilization were delumped through a Waring blender thusproducing a fine particulate suspension entering the fermenter. Thetransformation growth media, canrenone addition schedules, nutrientaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Tables 22 and 23.

                                      TABLE 22                                    __________________________________________________________________________    Descriptions of Process for Canrenone Bioconversion                           Run Number                                                                             R3A      R3B     R3C      R3D                                        __________________________________________________________________________    Medium (g/L)                                                                    Corn steep liq. 30 the same as run Peptone: 20 the same as run                Yeast extract 15 R3A Yeast Ext.: 20 R3A                                       NH.sub.4 H.sub.2 PO.sub.4 3  Glucose: 20                                      Glucose 15  OSA: 3 ml                                                         OSA 0.5 ml                                                                    pH adjusted to 6.5  adjusted to 6.5                                            with 2.5N NaOH  with 2.5N NaOH                                               Canrenone charge canrenone was the same as run Non-sterile The same as                                         run                                          at sterilized and R3A canrenone: R3A                                           blended. BI: 50 g BI: 50 g charged by the                                     16.5 hrs: 110 g 16.5 hrs: 110 g scheduled listed BI: 50 g                      46.5 hrs: 80 g in Table 23 16.5 hrs: 110 g                                  Feedings see Table 23 see Table 23 see Table 23 see Table 23                  Harvest time 118.5 hrs. 118.5 hrs. 118.5 hrs. 73.5 hrs.                       Bioconversion 93.7% 94.7% 60.0% 68.0%                                       __________________________________________________________________________

                                      TABLE 23                                    __________________________________________________________________________    The Feeding Schedule for Canrenone, Glucose and                                 Growth Medium in the Development Experiment                                       R3C                         R3A    R3B    R3D                                              Peptone &                                                                           Antibiotics                                                                            Canrenone/                                                                           Canrenone/                                                                           Canrenone/                       canrenone  Yeast ext. 20 mg kanamycin Growth Growth Growth                    200 g/2 L  20 g each 20 mg Medium Medium Medium                               sterile Glucose 50% in IL tetracycline see Table see Table see Table                                                        Addition DI solution                                                         water 100 mg cefalexin 22                                                     22 22                           Time hr. g g g in 50 ml g/L g/L g/L                                         __________________________________________________________________________    0     --    --     --    --        50 g/0.4 L                                                                           50 g/0.4 L                                                                           50 g/0.4 L                     14.5 16 100 25 50 ml -- -- --                                                 16.5 -- -- -- -- 110 g/1.2 L 110 g/1.2 L 110 g/1.2 L                          20.5 16 140 25 -- -- -- --                                                    28.5 16 140 25 -- -- -- --                                                    34.5 16 150 25 -- -- -- --                                                    40.5 16 150 25 50 ml -- -- --                                                 46.5 880  130 25 -- --  80 g/0.8 L --                                         52.5 160  120 25 -- -- -- --                                                  58.5 160  150 25 -- -- -- --                                                  64.5 160  180 25 50 ml -- -- --                                               70.5 160  140 25 -- -- -- --                                                __________________________________________________________________________

Due to filamentous growth, a highly viscous fermenter broth was seen inall four of the runs of this Example. To overcome obstacles which highviscosity created with respect to aeration, mixing, pH control andtemperature control, the aeration rate and agitation speed wereincreased during these runs. Conversions proceeded satisfactorily underthe more severe conditions, but a dense cake formed above the liquidbroth surface. Some unreacted canrenone was carried out of the broth bythis cake.

EXAMPLE 8

The description and results of Example 8 are summarized in Table 24.Four fermentation runs were made in which 11α-hydroxycanrenone wasproduced by bioconversion of canrenone. In two of these runs (R4A andR4D), the bioconversion was conducted in substantially the same manneras runs R3A and R3D of Example 6. In run R4C, canrenone was converted to11α-hydroxycanrenone generally in the manner described in Example 3. InRun R4B, the process was carried out generally as described in Example4, i.e., with sterilization of canrenone and growth medium in thefermenter just prior to inoculation, all nitrogen and phosphorusnutrients were introduced at the start of the batch, and a supplementalsolution containing glucose only was fed into the fermenter to maintainthe glucose level as the batch proceeded. In the latter process (runR4B), glucose concentration was monitored every 6 hours and glucosesolution added as indicated to control glucose levels in the 0.5 to 1%range. The canrenone addition schedules for these runs are set forth inTable 25.

                                      TABLE 24                                    __________________________________________________________________________    Descriptions of the Process                                                     Development Experiment of Canrenone Bioconversions                          Run Number                                                                              R4A      R4B       R4C      R4D                                     __________________________________________________________________________    Medium (g/L)                                                                    Corn steep liq. 30 the same as run Peptone: 20 the same as run                Yeast extract 15 R4A Yeast ext.: 20 R4A                                       NH4H2PO4 3  Glucose: 20                                                       Glucose 15  OSA 3 ml                                                          OSA 0.5 ml                                                                    pH adjusted to 6.5  adjusted to 6.5                                            with 2.5N NaOH  with 2.5N NaOH                                               Canrenone charge at Canrenone was 160 g canrenone is Nonsterile                                                   Canrenone was                              sterilized and sterilized in the canrenone: sterilized and                    blended. fermenter charged by the blended.                                    BI: 40 g  schedule listed BI: 40 g                                            23.5 hrs: 120 g  in Table 25 23.5 hrs: 120 g                                 Medium charge see Table 25 see Table 25 see Table 25 see Table 25                                                  Harvest time 122 hrs. 122 hrs. 122                                           hrs. 122 hrs.                             Bioconversion 95.6% 97.6% 95.4% 96.7%                                       __________________________________________________________________________

                                      TABLE 25                                    __________________________________________________________________________    The Feeding Schedule of Canrenone, Glucose and                                  Growth Medium in the Development Experiment                                       R4C                                                                                            Antibiotics                                                  20 mg kanamycin                                                              Peptone & 20 mg                                                             Canrenone  Yeast ext. tetracycline R4A R4B R4D                                200 g/2 L Glucose 20 g each 100 mg cefalexin Growth Growth Growth                                                         sterile 50% in 1 L in 50                                                    ml (added Medium Medium                                                       Medium                             Addition water solution water in canrenone see Table see Table see                                                       Table                              Time hr. g g g slurry) 24 24 24                                             __________________________________________________________________________    14    600   135  25    50 ml    --     --    --                                 20 -- 100 -- -- -- -- --                                                      23 -- -- -- -- 120 g/1.2 L -- 120 g/1.2 L                                     26 -- 100 25 -- -- -- --                                                      32 -- 135 25 -- -- -- --                                                      38 500 120 25 50 ml -- -- --                                                  44 -- 100 25 -- -- -- --                                                      50 -- 100 25 -- -- -- --                                                      56 -- 150 25 -- -- -- --                                                      62 500 150 25 50 ml -- -- --                                                  68 -- 200 25 -- -- -- --                                                      74 -- 300 25 -- -- -- --                                                      8- -- 100 25 -- -- -- --                                                      86 -- 125 25 -- -- -- --                                                      92 -- 175 25 -- -- -- --                                                      98 -- 150 -- -- -- -- --                                                      104  -- 175 -- -- -- -- --                                                    110  -- 175 -- -- -- -- --                                                    116  -- 200 -- -- -- -- --                                                  __________________________________________________________________________

All fermenters were run under high agitation and aeration during most ofthe fermentation cycle because the fermentation beer had become highlyviscous within a day or so after inoculation.

EXAMPLE 9

The transformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 26.

Four bioconversion runs were carried out substantially in the mannerdescribed for run R4B of Example 8, except as described below. In runR5B, the top turbine disk impeller used for agitation in the other runswas replaced with a downward pumping marine impeller. The downwardpumping action axially poured the broth into the center of the fermenterand reduced cake formation. Methanol (200 ml) was added immediatelyafter inoculation in run R5D. Since canrenone was sterilized in thefermenter, all nutrients except glucose were added at the start of thebatch, obviating the need for chain feeding of sources of nitrogen,sources of phosphorus or antibiotics.

                                      TABLE 26                                    __________________________________________________________________________    Process Description of the Process                                              Development Experiment of 10 L Scale Bioconversions                         Run Number                                                                             R5A      R5B      R5C      R5D                                       __________________________________________________________________________    Medium (g/L)                                                                    Corn steep liq. 30 the same as run Peptone: 20 the same as run                Yeast Extract 15 R5A Yeast Ext.: 20 R5A                                       NH.sub.4 H.sub.2 PO.sub.4 3  Glucose: 20                                      Glucose 15  OSA 3 ml                                                          OSA 0.5 ml                                                                    pH adjusted to 6.5  adjusted to 6.5                                            with 2.5N NaOH  with 2.5N NaOH                                               Canrenone charge 160 g canrenone 160 g canrenone 160 g canrenone 160 g                                          canrenone                                    sterilized in the sterilized in the sterilized in sterilized in                                                  fermenter fermenter the fermenter                                           the fermenter                               Medium feeding glucose feeding glucose feeding glucose feeding glucose                                          feeding                                     Harvest time 119.5 hrs. 119.5 hrs. 106 119.5 hrs.                             Bioconversion 96% 94.1% 88.5% 92.4%                                         __________________________________________________________________________

In order to maintain immersion of the solid phase growing above theliquid surface, growth medium (2 L) was added to each fermenter 96 hoursafter the beginning of the batch. Mixing problems were not entirelyovercome by either addition of growth medium or use of a downwardpumping impeller (run R5B) but the results of the runs demonstrated thefeasibility and advantages of the process, and indicated thatsatisfactory mixing could be provided according to conventionalpractices.

EXAMPLE 10

Three bioconversion runs were carried out substantially in the mannerdescribed in Example 9. The transformation growth media, canrenoneaddition schedules, harvest times, and degrees of conversion for theruns of this Example are set forth in Table 27:

                  TABLE 27                                                        ______________________________________                                        Process Description of the                                                      Experiment 10 L Scale Bioconversion                                           Run Number R6A         R6B       R6C                                        ______________________________________                                        Medium (g/L)                                                                    Corn steep liq. 30 the same as run Peptone: 20                                Yeast Extract 15 R6A Yeast Ext.: 20                                           NH.sub.4 H.sub.2 PO.sub.4 3  Glucose: 20                                      Glucose 15  OSA                                                               OSA 0.5 ml  0.5 ml                                                            pH adjusted to 6.5  adjusted to 6.5                                            with 2.5N NaOH  with 2.5N NaOH                                               Canrenone 160 g canrenone 160 g canrenone 160 g canrenone                     charge sterilized in the sterilized in the sterilized in                       fermenter fermenter the fermenter                                            Medium glucose feeding; glucose feeding; glucose                              feeding 1.3 L medium 0.5 L medium feeding; no                                  and 0.8 L sterile and 0.5 L sterile other addition                            water at 71 hrs. water at 95 hrs                                             Harvest time 120 hrs. 120 hrs. 120 hrs.                                       Bioconversion 95% 96% 90%                                                     Mass Balance 59% 54% 80%                                                    ______________________________________                                    

Growth medium (1.3 L) and sterile water (0.8 L) were added after 71hours in run R6A to submerge mycelial cake which had grown above thesurface of the liquid broth. For the same purpose, growth medium (0.5 L)and sterile water (0.5 L) were added after 95 hours in run R6B. Materialbalance data showed that a better mass balance could be determined wherecake buildup above the liquid surface was minimized

EXAMPLE 11

Fermentation runs were made to compare pre-sterilization of canrenonewith sterilization of canrenone and growth medium in the transformationfermenter. In run R7A, the process was carried out as illustrated inFIG. 2, under conditions comparable to those of runs R2C, R2D, R3A, R3B,R3D, R4A, and R4D. Run R7B was as illustrated in FIG. 3 under conditionscomparable to those of Examples 4, 9 and 10, and run R4B. Thetransformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 28:

                  TABLE 28                                                        ______________________________________                                        Process Description of the                                                      Experiment of 10 L Scale Bioconversions                                         Run Number   R7A           R7B                                            ______________________________________                                        Medium (g/L)                                                                    corn steep liq. 30 the same as run                                            Yeast extract 15 R7A                                                          NH.sub.4 H.sub.2 PO.sub.4 3                                                   Glucose 15                                                                    OSA 0.5 ml                                                                    pH adjusted to 6.5                                                             with 2.5N NaOH                                                               Canrenone charge 160 g canrenone 160 g canrenone                               was sterilized & was sterilized                                               blended outside in the fermenter                                              the fermenter                                                                Medium charge Glucose feeding; Glucose feeding;                                canrenone was no other                                                        added with 1.6 L addition                                                     growth medium                                                                Harvest time 118.5 hrs. 118.5 hrs.                                            Bioconversion 93% 89%                                                       ______________________________________                                    

A mass balance based on the final sample taken from run R7B was 89.5%,indicating that no significant substrate loss or degradation inbioconversion. Mixing was determined to be adequate for both runs.

Residual glucose concentration was above the desired 5-10 gpl controlrange during the initial 80 hours. Run performance was apparentlyunaffected by a light cake that accumulated in the head space of boththe fermenters.

EXAMPLE 12

Extraction efficiency was determined in a series of 1 L extraction runsas summarized in Table 29. In each of these runs, steroids wereextracted from the mycelium using ethyl acetate (1 L/L fermentationvolume). Two sequential extractions were performed in each run. Based onRP-HPLC, About 80% of the total steroid was recovered in the firstextraction; and recovery was increased to 95% by the second extraction.A third extraction would have recovered another 3% of steroid. Theremaining 2% is lost in the supernatant aqueous phase. The extract wasdrawn to dryness using vacuum but was not washed with any additionalsolvent. Chasing with solvent would improve recovery from the initialextraction if justified by process economics.

                  TABLE 29                                                        ______________________________________                                        Recovery of 11α-Hydroxycanrenone                                          at 1 Liter Extraction (% of Total)                                              Run      1st     2nd      3rd                                               Number Extract Extract Extract Supernatant                                  ______________________________________                                        R5A      79%     16%        2%    2%                                            R5A 84% 12% 2% 2%                                                             R4A 72% 20% 4% 4%                                                             R4A 79% 14% 2% 5%                                                             R4B 76% 19% 4% 1%                                                             R4B 79% 16% 3% 2%                                                             R4B 82% 15% 2% 1%                                                             Average 79% 16% 3% 2%                                                       ______________________________________                                    

Methyl isobutyl ketone (MIBK) and toluene were evaluated asextraction/crystallization solvents for 11α-hydroxycanrenone at the 1 Lbroth scale. Using the extraction protocol as described hereinabove,both MIBK and toluene were comparable to ethyl acetate in bothextraction efficiency and crystallization performance.

EXAMPLE 13

As part of the evaluation of the processes of FIGS. 2 and 3, particlesize studies were conducted on the canrenone substrate provided at thestart of the fermentation cycle in each of these processes. As describedabove, canrenone fed to the process of FIG. 1 was micronized beforeintroduction into the fermenter. In this process, the canrenone is notsterilized, growth of unwanted microorganisms being controlled byaddition of antibiotics. The processes of FIGS. 2 and 3 sterilize thecanrenone before the reaction. In the process of FIG. 2, this isaccomplished in a blender before introduction of canrenone into thefermenter. In the process of FIG. 3, a suspension of canrenone in growthmedium is sterilized in the fermenter at the start of the batch. Asdiscussed hereinabove, sterilization tends to cause agglomeration ofcanrenone particles. Because of the limited solubility of canrenone inthe aqueous growth medium, the productivity of the process depends onmass transfer from the solid phase, and thus may be expected to dependon the interfacial area presented by the solid particulate substratewhich in turn depends on the particle size distribution. Theseconsiderations initially served as deterrents to the processes of FIGS.2 and 3.

However, agitation in the blender of FIG. 2 and the fermentation tank ofFIG. 3, together with the action of the shear pump used for transfer ofthe batch in FIG. 2, were found to degrade the agglomerates to aparticle size range reasonably approximate that of the unsterilized andmicronized canrenone fed to the process of FIG. 1. This is illustratedby the particle size distributions for the canrenone as available at theoutset of the reaction cycle in each of the three processes. See Table30 and FIGS. 4 and 5.

                  TABLE 30                                                        ______________________________________                                        Particle Distributions of                                                       Three Different Canrenone Samples                                                                         mean                                                 size Run #: %                                                              Sample 45-125μ <180μ μ Bioconversion                               ______________________________________                                        Canrenone                                                                                75%        95%   --     R3C:                                         shipment    93.1% (120 h)                                                         R4C:                                                                          96.3% (120 h)                                                             Blended 31.2% 77.2% 139.5 R3A:                                                Sample    94.6% (120 h)                                                           R3B:                                                                          95.2% (120 h)                                                             Sterilized 24.7% 65.1% 157.4 R4B:                                             Sample    97.6% (120 h)                                                           R5B:                                                                          93.8% (120 h)                                                           ______________________________________                                    

From the data in Table 30, it will be noted that agitators and shearpump were effective to reduce the average particle size of thesterilized canrenone to the same order of magnitude as the unsterilizedsubstrate, but a significance size difference remained in favor of theunsterilized substrate. Despite this difference, reaction performancedata showed that the pre-sterilization processes were at least asproductive as the process of FIG. 1. Further advantages may be realizedin the process of FIG. 2 by certain steps for further reducing andcontrolling particle size, e.g., wet milling of sterilized canrenone,and/or by pasteurizing rather than sterilizing.

EXAMPLE 14

A seed culture was prepared in the manner described in Example 5. At 20hours, the mycelia in the inoculum fermenter was pulpy with a 40% PMV.Its pH was 5.4 and 14.8 gpl glucose remained unused.

A transformation growth medium (35 L) was prepared having thecomposition shown in Table 20. In the preparation of feeding medium,glucose and yeast extract were sterilized separately and mixed as asingle feed at an initial concentration of 30% by weight glucose and 10%by weight yeast extract. pH of the feed was adjusted to 5.7.

Using this medium, (Table 20), two bioconversion runs were made for theconversion of canrenone to 11α-hydroxycanrenone. Each of the runs wasconducted in a 60 L fermenter provided with an agitator comprising oneRushton turbine impeller and two Lightnin' A315 impellers.

Initial charge of the growth medium to the fermenter was 35 L.Micronized and unsterilized canrenone was added to an initialconcentration of 0.5%. The medium in the fermenter was inoculated with aseed culture prepared in the manner described in Example 5 at an initialinoculation ratio of 2.5%. Fermentation was carried out at a temperatureof 28° C., an agitation rate of 200 to 500 rpm, an aeration rate of 0.5vvm, and backpressure sufficient to maintain a dissolved oxygen level ofat least 20% by volume, The transformation culture developed during theproduction run was in the form of very small oval pellets (about 1-2mm). Canrenone and supplemental nutrients were chain fed to thefermenter generally in the manner described in Example 1. Nutrientadditions were made every four hours at a ratio of 3.4 g glucose and 0.6g yeast extract per liter of broth in the fermenter.

Set forth in Table 31 are the aeration rate, agitation rate, dissolvedoxygen, PMV, and pH prevailing at stated intervals during each of theruns of this Example, as well as the glucose additions made during thebatch. Table 32 shows the canrenone conversion profile. Run R11A wasterminated after 46 hours; Run R11B continued for 96 hours. In thelatter run, 93% conversion was reached at 81 hours; one more feedaddition was made at 84 hours; and feeding then terminated. Note that asignificant change in viscosity occurred between the time feeding wasstopped and the end of the run.

                  TABLE 31                                                        ______________________________________                                        December 11, 1996                                                               153SRL 6029                                                                   PATENT                                                                               air                                   Gluc                             Time (1 pm) rpm % DO Backpress PMV (%) pH cc (g/l)                          ______________________________________                                        Fermentation R11A                                                               0.1    20      200  93    0      2      6.17 5.8                              7 20 200 85.1 0 5 6.03 5.5                                                    12.4 20 300 50.2 0  5.43                                                      21.8 20 400 25.5 0 38 6.98 0                                                  29 20 500 17 0 35 5.22                                                        30.2 20 500 18.8 10  5.01                                                     31 20 500 79 10  4.81 1                                                       35.7 20 500 100 10 45 5.57 0                                                  46.2 20 500 23 6 45 5.8 1                                                   ______________________________________                                        Total glucose: 27.5 g/l                                                         Total yeast extract: 8.75 g/l                                               ______________________________________                                          Fermentation R11B                                                                 0.1    20    200  92.9  0      2      5.98 5.4                            7 20 200 82.3 0 5 5.9 5                                                       12.4 20 300 49.5 0  5.48                                                      21.8 20 400 18 0 40 7.12 0                                                    29 20 500 36.8 0 35 5.1 3                                                     35.7 20 500 94.5 10  4.74 0                                                   46.2 20 500 14.5 6 45 5.32 2                                                  55 20 500 16.7 10  5.31 0.5                                                   58.6 20 500 19.4 15  5.32 1                                                   61.9 20 500 13 15 40 5.36 2                                                   71.7 20 500 13 15 42 5.37 0                                                   81.1 20 500 22.9 15  5.42 2.5                                                 85.6 20 500 22 15 45 5.48 1                                                   97.5 20 500 108 15 45 6.47 0                                                  117.7 20 500  15  7.38 0                                                    ______________________________________                                        Total glucose: 63 g/l                                                           Total yeast extract: 14.5 g/l                                               ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        December 11, 1996                                                               155SRL 6029                                                                   PATENT                                                                                                          Conv.                                       Concentrations (g/l) Con- Calc rates (g/l/h)                                             OH-    Can-      version                                                                             OH-can                                                                              Cal-  Mea-                          Sample                                                                              Time   can    ren. Total                                                                              (%)   (g/l) culated                                                                             sured                         ______________________________________                                        Fermentation R11A: Canrenone conversion                                         R11A-0  0.10   0.00 5.41 5.41                                                 R11A-7 7.00 0.18 4.89 5.07 3.58 0.18 0.03 0.03                                R11A- 21.80 2.02 2.12 4.14 48.75 2.44 0.15 0.12                               22                                                                            R11A- 29.00 3.67 4.14 7.81 47.03 4.48 0.28 0.23                               29                                                                            R11A- 35.70 6.68 1.44 8.12 82.27 7.74 0.49 0.45                               36                                                                            R11A- 46.20 7.09 0.41 7.51 94.48 8.59 0.08 0.04                               46                                                                          ______________________________________                                        Fermentation R11B: Canrenone conversion                                         R11B-0  0.1    0.00 5.60 5.60                                                 R11B-7 7.0 0.20 4.98 5.18 3.78 0.19 0.03 0.03                                 R11B- 21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16                                22                                                                            R11B- 29.0 4.48 16.99 21.47 20.87 4.69 0.30 0.27                              29                                                                            R11B- 35.7 8.18 10.35 18.53 44.16 9.70 0.75 0.55                              36                                                                            R11B- 55.0 17.03 13.20 30.23 56.33 19.50 0.32 0.36                            55                                                                            R11B- 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05                            59                                                                            R11B- 61.9 22.19 8.62 30.81 72.02 24.50 0.77 0.42                             62                                                                            R11B- 71.7 26.62 3.61 30.23 88.06 29.46 0.51 0.45                             72                                                                            R11B- 81.1 27.13 2.05 29.18 92.97 30.32 0.09 0.05                             81                                                                            R11B- 85.6 26.87 2.02 28.88 93.02 30.11 -0.04 -0.06                           86                                                                            R11B- 97.5 23.95 1.71 25.66 93.34 30.22 0.01 -0.25                            97                                                                            R11B- 117.7 24.10 1.68 25.79 93.47 30.26 0.00 0.01                            118                                                                         ______________________________________                                    

EXAMPLE 15

Various cultures were tested for effectiveness in the bioconversion ofcanrenone to 11α-canrenone according to the methods generally describedabove.

A working cell bank of each of Aspergillus niger ATCC 11394, Rhizopusarrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b was prepared inthe manner described in Example 5. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 18. Each culture was used for inoculation of two flasks, a totalof six. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36° C.,and a 0.5 ml aliquot of this solution was introduced into each of theflasks. Bioconversion was carried out generally under the conditionsdescribed in Example 5 with additions of 50% by weight glucose solution(1 ml) each day. After the first 72 hours the following observationswere made on the development of mycelia in the respective transformationfermentation flasks:

ATCC 11394--good even growth

ATCC 11145--good growth in first 48 hours, but mycelial clumped into aball; no apparent growth in last 24 hours;

ATCC 6227b--good growth; mycelial mass forming clumped ball.

Samples of the broth were taken to analyze for the extent ofbioconversion. After three days, the fermentation using ATCC 11394provided conversion to 11α-hydroxycanrenone of 80 to 90%; ATCC 11145provided a conversion of 50%; and ATCC 6227b provided a conversion of 80to 90%.

EXAMPLE 16

Using the substantially the method described in Example 15, theadditional microorganisms were tested for effectiveness in theconversion of canrenone to 11α-hydroxycanrenone. The organisms testedand the results of the tests are set forth in Table 33:

                  TABLE 33                                                        ______________________________________                                        December 11, 1996                                                               159SRL 6029                                                                   PATENT                                                                        Cultures tested for Bioconversion                                             of canrenone to 11 alpha-hydroxy-canrenone                                                                        approximate                               Culture ATTC # media.sup.1 results conversion                               ______________________________________                                        Rhizopus oryzae                                                                           1145      CSL     +     50%   -                                     Rhizopus stolonifer 6227b CSL + 80-90% -                                      Aspergillus nidulans 11267 CSL + 50% 80%                                      Aspergillus niger 11394 CSL + 80-90% -                                        Aspergillus ochraceus NRRL 405 CSL +  90%                                     Aspergillus ochraceus 18500 CSL +  90%                                        Bacillus subtilis 31028 P&CSL - 0% 0%                                         Bacillus subtilis 31028 CSL - 0% 0%                                           Bacillus sp. 31029 P&CSL - 0% 0%                                              Bacillus sp. 31029 CSL - 0% *                                                 Bacillus megaterium 14945 P&CSL + 5% 80%*                                     Bacillus megaterium 14945 CSL + 5% 10%*                                       Trichothecium roseum 12519 CSL + 80%* 90%*                                    Trichothecium roseum 8685 CSL + 80%* 90%*                                     Streptomyces fradiae 10745 CSL + <5% <10%                                     Streptomyces fradiae 10745 TSB - * *                                          Streptomyces 13664 CSL - 0% *                                                 lavendulae                                                                    Streptomyces 13664 TSB - 0% 0%                                                lavendulae                                                                    Nocardiodes simplex 6946 BP - 0% 0%                                           Nocardiodes simplex 13260 BP - * *                                            Pseudomonas sp. 14696 BP - * *                                                Pseudomonas sp. 14696 CSL + <5% <10%                                          Pseudomonas sp. 14696 TSB - 0% *                                              Pseudomonas sp. 13261 BP + * <10%                                             Pseudomonas 13262 BP  # <10%                                                  cruciviae                                                                     Pseudomonas putida 15175 BP - 0% 0%                                           *formation of other                                                           unidentified products                                                       ______________________________________                                         .sup.1 Media: CSL  corn steep liquor; TSB  tryptic soy broth; P&CSL           peptone and acorn steep liquor; BP  beef extract and peptone.            

EXAMPLE 17

Various microorganisms were tested for effectiveness in the conversionof canrenone to 9α-hydroxycanrenone. Fermentation media for the runs ofthis Example were prepared as set forth in Table 34:

                  TABLE 34                                                        ______________________________________                                        Soybean Meal:                                                                   dextrose 20 g                                                                 soybean meal 5 g                                                              NaCl 5 g                                                                      yeast extract 5 g                                                             KH.sub.2 PO.sub.4 5 g                                                         water to 1 L                                                                  pH 7.0                                                                        Peptone/yeast extract/glucose:                                                glucose 40 g                                                                  bactopeptone 10 g                                                             yeast extract 5 g                                                             water to 1 L                                                                  Mueller-Hinton:                                                               beef infusion 300 g                                                           casamino acids 17.5 g                                                         starch 1.5 g                                                                  water to 1 L                                                                ______________________________________                                    

Fungi were grown in soybean meal medium and in peptone-yeast extractglucose; atinomycetes and eubacteria were grown in soybean meal (plus0.9% by weight Na formate for biotransformations) and in Mueller-Hintonbroth.

Starter cultures were inoculated with frozen spore stocks (20 ml soybeanmeal in 250 ml Erlenmayer flask). The flasks were covered with a milkfilter and bioshield. Starter cultures (24 or 48 hours old) were used toinoculate metabolism cultures (also 20 ml in 250 ml Erlenmeyerflask)--with a 10% to 15% crossing volume--and the latter incubated for24 to 48 hours before addition of steroid substrate for thetransformation reaction.

Canrenone was dissolved/suspended in methanol (20 mg/ml), filtersterilized, and added to the cultures to a final concentration of 0.1mg/ml. All transformation fermentation flasks were shaken at 250 rpm (2"throw) in a controlled temperature room at 26° C. and 60% humidity.

Biotransformations were harvested at 5 and 48 hours, or at 24 hours,after addition of substrate. Harvesting began with the addition of ethylacetate (23 ml) or methylene chloride to the fermentation flask. Theflasks were then shaken for two minutes and the contents of each flaskpoured into a 50 ml conical tube. To separate the phases, tubes werecentrifuged at 4000 rpm for 20 minutes in a room temperature unit. Theorganic layer from each tube was transferred to a 20 ml borosilicateglass vial and evaporated in a speed vac. Vials were capped and storedat -20° C.

To obtain material for structure determination, biotransformations werescaled up to 500 ml by increasing the number of shake flaskfermentations to 25. At the time of harvest (24 or 48 hours afteraddition of substrate), ethyl acetate was added to each flaskindividually, and the flasks were capped and put back on the shaker for20 minutes. The contents of the flasks were then poured intopolypropylene bottles and centrifuged to separate the phases, or into aseparatory funnel in which phases were allowed to separate by gravity.The organic phase was dried, yielding crude extract of steroidscontained in the reaction mixture.

Reaction product was analyzed first by thin layer chromatography onsilica gel (250 μm) fluorescence backed plates (254 nm). Ethyl acetate(500 μL was added to each vial containing dried ethyl acetate extractfrom the reaction mixture. Further analyses were conducted by highperformance liquid chromatography and mass spectrometry. Plates weredeveloped in a 95:5 v/v chloroform/methanol medium.

Further analysis was conducted by high performance liquid chromatographyand mass spectrometry. A waters HPLC with Millennium software,photodiode array detector and autosampler was used. Reversed phase HPLCused a waters NovaPak C-18 (4 μm particle size) RadialPak 4 mmcartridge.The 25 minute linear solvent gradient began with the column initializedin water:acetonitrile (75:25), and ended at water-acetonitrile (25:75).This was followed by a three minute gradient to 100% acetonitrile and 4minutes of isocratic wash before column regeneration in initialconditions.

For LC/MS, ammonium acetate was added to both the acetonitrile and waterphases at a concentration of 2 nM. Chromatography was not significantlyaffected. Eluant from the column was split 22:1, with the majority ofthe material directed to the PDA detector. The remaining 4.5% of thematerial was directed to the electrospray ionizing chamber of an SciexAPI III mass spectrometer. Mass spectrometry was accomplished inpositive mode. An analog data line from the PDA detector on the HPLCtransferred a single wave length chromatogram to the mass spectrometerfor coanalysis of the UV and MS data.

Mass spectrometric fragmentation patterns proved useful in sorting fromamong the hydroxylated substrates. The two expected hydroxylatedcanrenones, 11α-hydroxy- and 9α-hydroxy, lost water at differentfrequencies in a consistent manner which could be used as a diagnostic.Also, the 9α-hydroxycanrenone formed an ammonium adduct more readilythan did 11α-hydroxycanrenone. Set forth in Table 35 is a summary of theTLC, HPLC/UV and LC/MS data for canrenone fermentations, showing whichof the tested microorganism were effective in the bioconversion ofcanrenone to 9α-hydroxycanrenone. Of these, the preferred microorganismwas Corynespora cassiicola ATCC 16718.

                  TABLE 35                                                        ______________________________________                                        Summary of TLC, HPLC/UV, and LC/MS                                              Data for Canrenone Fermentations                                                           Evidence for 9αOH-canrenone                                                       HPLC-    MS: 357                                        TLC peak at (M + H,                                                           spot at 9αQH- 339 (-H.sub.2 O)                                          9αQH- canrenone & 375                                                  Culture AD w/UV (+NH.sub.4)                                                 ______________________________________                                        Absidia coerula ATCC                                                                         n         y        y/n                                           6647                                                                          Absidia glauca ATCC n                                                         22752                                                                         Actinomucor elegans ATCC tr y tr                                              6476                                                                          Aspergilus flavipes tr                                                        ATCC 1030                                                                     Aspergillus fumigatus tr y n                                                  ATCC 26934                                                                    Aspergillus nidulans tr y y                                                   ATCC 11267                                                                    Aspergillus niger ATCC n y y                                                  16888                                                                         Aspergillus niger ATCC n y n                                                  26693                                                                         Aspergillus ochraceus n y n                                                   ATCC 18500                                                                    Bacterium cyclo-oxydans n tr n                                                (Searle) ATCC 12673                                                           Beauveria bassiana ATCC tr y y                                                7159                                                                          Beauveria bassiana ATCC y y y                                                 13144                                                                         Botryosphaeria obtusa y tr tr                                                 IMI 038560                                                                    Calonectria decora ATCC n tr y                                                14767                                                                         Chaetomium cochliodes tr tr y/n                                               ATCC 10195                                                                    Comomonas testosteroni tr tr n                                                (Searle) ATCC 11996                                                           Corynespora cassiicola y y y                                                  ATCC 16718                                                                    Cunninghamella y y y                                                          blakesleana ATCC 8688a                                                        Cunninghamella y y y                                                          echinulata ATCC 3655                                                          Cunninghamella elegans y y y                                                  ATCC 9245                                                                     Curcularia clavata ATCC n y y/n                                               22921                                                                         Curvularia lunata ATCC y n n                                                  12071                                                                         Cylindrocarpon tr n n                                                         radicicola (Searle) ATCC                                                      11011                                                                         Epicoccum humucola ATCC y y y                                                 12722                                                                         Epicoccum oryzae ATCC tr tr tr                                                12724                                                                         Fusarium oxysporum ATCC tr                                                    7601                                                                          Fusarium oxysporum f. sp. n                                                   cepae ATCC 11171                                                              Gibberella fujikuroi tr y y                                                   ATCC 14842                                                                    Gliocladium deliguescens y tr tr                                              ATCC 10097                                                                    Gongronella butieri ATCC y y UV?  y                                           22822                                                                         Hypomces chrysosoermus y y y                                                  Tul. IMI 109891                                                               Lipomyces lipofer ATCC n                                                      10792                                                                         Melanospora ornata ATCC tr n n                                                26180                                                                         Mortierella isabellinay y y n                                                 ATCC 42613                                                                    Mucor grisco-cyanus ATCC n                                                    1207a                                                                         Mucor mucedo ATCC 4605 tr y y                                                 Mycobacterium fortuitumn                                                      ATCC 6842                                                                     Myrothecium verrucaria tr tr y                                                ATCC 9095                                                                     Nocardia aurentia n tr n                                                      (Searle) ATCC 12674                                                           Nocardia cancicruria y y n                                                    (Searle)                                                                      Nocardia corallina ATCC n                                                     19070                                                                         Paecilonyces carneus n y n                                                    ATCC 46579                                                                    Penicillium chrysogenum n                                                     ATCC 9480                                                                     Penicillium patulum ATCC y y y/n                                              24550                                                                         Penicillium purpurogenum tr y y                                               ATCC 46581                                                                    Pithomyces atro- tr y tr                                                      olivaceus ATCC 6651                                                           Pithomyces cynodontis n tr tr                                                 ATCC 26150                                                                    Phycomyces blakesleeanus y y y/n                                              Pycnosporium sp. ATCC y y y/n                                                 12231                                                                         Rhizopogon sp.                                                                Rhizopus arrhizus ATCC tr y n                                                 11145                                                                         Rhizopus stolonifer ATCC n                                                    6227b                                                                         Rhodococcus equi ATCC n tr n                                                  14887                                                                         Rhodococcus equi ATCC tr tr                                                   21329                                                                         Rhodococcus sp. n n n                                                         Rhodococcus rhodochrous n tr n                                                ATCC 19150                                                                    Saccharopolyspora y y y                                                       erythaea ATCC 11635                                                           Sepedonium ampullosporum n n n                                                IMI 203033                                                                    Sepedonium chrysospermum n                                                    ATCC 13378                                                                    Septomyxa affinis ATCC n y UV? y/n                                            6737                                                                          Stachylidium bicolor y y y/n                                                  ATCC 12672                                                                    Streptomyces n                                                                californicus ATCC 15436                                                       Streptomyces n                                                                cinereocrocatus ATCC                                                          3443                                                                          Streptomyces coelicolor n                                                     ATCC 10147                                                                    Streptomyces flocculus                                                        ATCC 25453                                                                    Streptomyces fradiae n                                                        ATCC 10745                                                                    Streptomyces griseus n                                                        subsp. griseus ATCC                                                           13968                                                                         Streptomyces griseus n                                                        ATCC 11984                                                                    Streptomyces hydrogenans n                                                    ATCC 19631                                                                    Streptomyces y y y                                                            hygroscopicus ATCC 27438                                                      Streptomyces lavendulae n                                                     Panlab 105                                                                    Streptomyces n                                                                paucisnorogenes ATCC                                                          25489                                                                         Streptomyces n tr tr                                                          paurpurascens ATCC 25489                                                      Streptomyces                                                                  roseochromogenes ATCC                                                         13400                                                                         Streptomyces spectabilis n                                                    ATCC 27465                                                                    Stysanus microsporus                                                          ATCC 2833                                                                     Syncephalastrum n                                                             racemosum ATCC 18192                                                          Thamnidium elegans ATCC                                                       18191                                                                         Thamnostylum piriforme y tr y                                                 ATCC 8992                                                                     Thielavia terricolan   n                                                      ATCC 13807                                                                    Trichoderma viride ATCC n                                                     26802                                                                         Trichothecium roseum tr y y/n                                                 ATCC 12543                                                                    Verticillium theobromae y tr tr                                               ATCC 12474                                                                  ______________________________________                                    

EXAMPLE 18

Various cultures were tested for effectiveness in the bioconversion ofandrostendione to 11α-hydroxyandrostendione according to the methodsgenerally described above.

A working cell bank of each of Aspergillus ochraceus NRRL 405 (ATCC18500); Aspergillus niger ATCC 11394; Aspergillus nidulans ATCC 11267;Rhizopus oryzae ATCC 11145; Rhizopus stolonifer ATCC 6227b;Trichothecium roseum ATCC 12519 and ATCC 8685 was prepared essentiallyin the manner described in Example 4. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 15. Each culture was used for inoculation of two flasks, a totalof 16. Androstendione (300 mg) was dissolved in methanol (6 ml) at 36°C., and a 0.5 ml aliquot of this solution was introduced into each ofthe flasks. Bioconversion was carried out generally under the conditionsdescribed in Example 6 for 48 hours. After 48 hours samples of the brothwere pooled and extracted with ethyl acetate as in Example 17. The ethylacetate was concentrated by evaporation, and samples were analyzed bythin layer chromatography to determine whether a product having achromatographic mobility similar to that of 11α-hydroxy-androstendionestandard (Sigma Chemical Co., St. Louis) was present. The results areshown in Table 36. Positive results are indicated as "+".

                  TABLE 36                                                        ______________________________________                                        Bioconversion of androstendione to 11                                           alpha-hydroxy-androstendione                                                                                     TLG                                        Culture ATTC # media results                                                ______________________________________                                        Rhizopus oryzae                                                                             11145        CSL     +                                            Rhizopus stolonifer  6227b CSL +                                              Aspergillus nidulans 11267 CSL +                                              Aspergillus niger 11394 CSL +                                                 Aspergillus ochraceus NRRL 405 CSL +                                          Aspergilius ochraceus 18500 CSL +                                             Trichothecium roseum 12519 CSL +                                              Trichothecium roseum  8685 CSL +                                            ______________________________________                                    

The data in Table 36 demonstrate that each of listed cultures wascapable of producing a compound from androstendione having the same Rfvalue as that of the 11α-hydroxyandrostendione standard.

Aspergillus ochraceus NRRL 405 (ATCC 18500) was retested by the sameprocedure described above, and the culture products were isolated andpurified by normal phase silica gel column chromatography using methanolas the solvent. Fractions were analyzed by thin layer chromatography.TLC plates were Whatman K6F silica gel 60 Å, 10×20 size, 250μ thickness.The solvent system was methanol:chloroform, 5:95, v/v. The crystallizedproduct and 11α-hydroxyandrostendione standard were both analyzed byLC-MS and NMR spectroscopy. Both compounds yielded similar profiles andmolecular weights.

EXAMPLE 19

Various microorganisms were tested for effectiveness in the conversionof mexrenone to 11β-hydroxymexrenone. Fermentation media for thisexample were prepared as described in Table 34.

The fermentation conditions and analytical methods were the same asthose in Example 17. TLC plates and the solvent system were as describedin Example 18. The rationale for chromatographic analysis is as follows:11α-hydroxymexrenone and 11α-hydroxycanrenone have the samechromatographic mobility. 11α-hydroxycanrenone and 9α-hydroxycanrenoneexhibit the same mobility pattern as 11α-hydroxyandrostendione and11β-hydroxyandrostendione. Therefore, 11β-hydroxymexrenone should havethe same mobility as 9α-hydroxycanrenone. Therefore, compounds extractedfrom the growth media were run against 9α-hydroxycanrenone as astandard. The results are shown in Table 36.

                  TABLE 37                                                        ______________________________________                                        Summary of TLC Data for                                                         11β-hydroxymexrenone Formation                                           from Mexrenone                                                                                              Spot                                            Microorganism Medium.sup.1 Character.sup.2                                  ______________________________________                                        Absidia coerula ATCC 6647                                                                        M, S     strong                                              Aspergillus niger ATCC S, P faint (S)                                         16888  ? (P)                                                                  Beauveria bassiana ATCC P strong                                              7159                                                                          Beauveria bassiana ATCC S, P ?, ?                                             13144                                                                         Botryosphaeria obtusa IMI  faint                                              038560                                                                        Cunninghamella                                                                blakesleeana ATCC 8688a S, P strong                                           echinulata ATCC 3655 S, P strong                                              elegans ATCC 9245 S, P strong                                                 Curvularia lunata ATCC S strong                                               12017                                                                         Gongronella butleri ATCC S, P strong                                          22822                                                                         Penicillium patulum ATCC S, P strong                                          24550                                                                         Penicillium purpurogenum S, P strong                                          ATCC 46581                                                                    Pithomyces atro-olivaceus S, P faint                                          IFO 6651                                                                      Rhodococcus equi ATCC M faint                                                 14887                                                                         Saccharopolyspora erythaea M, SF faint                                        ATCC 11635                                                                    Streptomyces hygroscopicus M, SF strong                                       ATCC 27438                                                                    Streptomyces purpurascens M, SF faint                                         ATCC 25489                                                                    Thamnidium elegans ATCC S, P faint                                            18191                                                                         Thamnostylum piriforme S, P faint                                             ATCC 8992                                                                     Trichothecium roseum ATCC P, S faint (P)                                      12543  ? (S)                                                                ______________________________________                                         .sup.1 M = MuellerHinton  p = PYG (peptone/yeast extract/glucose)  S =        soybean meal  SF = soybean meal plus formate                                  .sup.2 ? = questionable difference from no substrate control             

These data suggest that the majority of the organisms listed in thistable produce a product similar or identical to 11β-hydroxymexrenonefrom mexrenone.

EXAMPLE 20 Scheme 1

Step 1

Preparation of5'R(5'α),7'β-20'-Aminohexadecahydro-11'β-hydroxy-10'a,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'α(5'H)-[7,4]metheno[4H[cyclopenta[a]phenanthrene]-5'-carbonitrile.

Into a 50 gallon glass-line reactor was charged 61.2 L (57.8 kg) of DMFfollowed by 23.5 Kg of 11-hydroxycanrenone 1 with stirring. To themixture was added 7.1 kg of lithium chloride. The mixture was stirredfor 20 minutes and 16.9 kg of acetone cyanohydrin was charged followedby 5.1 kg of triethylamine. The mixture was heated to 85° C. andmaintained at this temperature for 13-18 hours. After the reaction 353 Lof water was added followed by 5.6 kg of sodium bicarbonate. The mixturewas cooled to 0° C., transferred to a 200 gallon glass-lined reactorwith quenched with 130 kg of 6.7% sodium hypochlorite solution slowly.The product was filtered and washed with 3×40 L portions of water togive 21.4 kg of the product enamine. ##STR123##

EXAMPLE 21 Scheme 1

Step 2

Preparation of4'S(4'α),7'α-Hexadecahydro-11'α-hydroxy-10'β,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'β(2'H)-carbonitrile.

Into a 200 gallon glass-lined reactor was charged 50 kg of enamine 2,approximately 445 L of 0.8 N dilute hydrochloric acid and 75 L ofmethanol. The mixture was heated to 80° C. for 5 hours, cooled to 0° C.for 2 hours. The solid product was filtered to give 36.5 kg of dryproduct diketone. ##STR124##

EXAMPLE 22 Scheme 1

Step 3A

Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

A 4-neck 5-L bottom flask was equipped with mechanical stirrer, pressureequalizing addition funnel with nitrogen inlet tube, thermometer andcondenser with bubbler. The bubbler was connected via tygon tubing totwo 2-L traps, the first of which was empty and placed to preventback-suction of the material in the second trap (1 L of concentratedsodium hypochlorite solution) into the reaction vessel. The diketone 3(79.50 g; [weight not corrected for purity, which was 85% ]) was addedto the flask in 3 L methanol. A 25% methanolic sodium methoxide solution(64.83 g) was placed in the funnel and added dropwise, with stirringunder nitrogen, over a 10 minute period After the addition was complete,the orangish yellow reaction mixture was heated to reflux for 20 hours.After this period, 167 mL of 4 N HCl was added (Caution: HCN evolutionat this point!) dropwise through the addition funnel to the stillrefluxing reaction mixture. The reaction mixture lightened in color to apale golden orange. The condenser was then replaced with a take-off headand 1.5 L of methanol was removed by distillation while 1.5 L of waterwas simultaneously added to the flask through the funnel, in concertwith the distillation rate. The reaction mixture was cooled to ambienttemperature and extracted twice with 2.25 L aliquots of methylenechloride. The combined extracts were washed successively with 750 mLaliquots of cold saturated NaCl solution, 1N NaOH and again withsaturated NaCl. The organic layer was dried over sodium sulfateovernight, filtered and reduced in volume to ˜250 mL in vacuo. Toluene(300 mL) was added and the remaining methylene chloride was strippedunder reduced pressure, during which time the product began to form onthe walls of the flask as a white solid. The contents of the flask werecooled overnight and the solid was removed by filtration. It was washedwith 250 mL toluene and twice with 250 mL aliquots of ether and dried ona vacuum funnel to give 58.49 g of white solid was 97.3% pure by HPLC.On concentrating the mother liquor, an additional 6.76 g of 77.1% pureproduct was obtained. The total yield, adjusted for purity, was 78%.

EXAMPLE 23 Scheme 1

Step 3B

Conversion of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone toMethyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone.

A 5-L four neck flask was equipped as in the above example, except thatno trapping system was installed beyond the bubbler. A quantity of138.70 g of the hydroxyester was added to the flask, followed by 1425 mLmethylene chloride, with stirring under nitrogen. The reaction mixturewas cooled to ˜5° C. using a salt/ice bath. Methanesulfonyl chloride(51.15 g, 0.447 mole) was added rapidly, followed by the slow dropwiseaddition of triethylamine (54.37 g) in 225 mL methylene chloride.Addition, which required ˜30 minutes, was adjusted so that thetemperature of the reaction never rose about 5° C. Stirring wascontinued for 1 hour post-addition, and the reaction contents weretransferred to a 12-L separatory funnel, to which was added 2100 mLmethylene chloride. The solution was washed successively with 700 mLaliquots each of cold 1N HCl, 1N NaOH, and saturated aqueous NaClsolution. The aqueous washes were combined and back-extracted with 3500mL methylene chloride. All of the organic washes were combined in a 9-Ljug, to which was added 500 g neutral alumina, activity grade II, and500 g anhydrous sodium sulfate. The contents of the jug were mixed wellfor 30 minutes and filtered. The filtrate was taken to dryness in vacuoto give a gummy yellow foam. This was dissolved in 350 mL methylenechloride and 1800 mL ether was added dropwise with stirring. The rate ofaddition was adjusted so that about one-half of the ether was added over30 minutes. After about 750 mL had been added, the product began toseparate as a crystalline solid. The remaining ether was added in 10minutes. The solid was removed by filtration, and the filter cake waswashed with 2 L of ether and dried in a vacuum oven at 50° C. overnight,to give 144.61 g (88%) nearly white solid, m.p. 149°-150° C. Materialprepared in this fashion is typically 98-99% pure by HPLC (area %). Inone run, material having a melting point of 153°-153.5° C. was obtained,with a purity, as determined by HPLC area, of 99.5%.

EXAMPLE 24 Scheme 1

Step 3C

Method A:

Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

A 1-L four neck flask was equipped as in the second example. Formic acid(250 mL) and acetic anhydride (62 mL) were added to the flask withstirring under nitrogen. Potassium formate (6.17 g) was added and thereaction mixture was heated with an oil bath to an internal temperatureof 40° C. (this was later repeated at 70° C. with better results) for 16hours. After 16 hours, the mesylate 5 was added and the internaltemperature was increased to 100° C. Heating and stirring were continuedfor 2 hours, after which the solvent was removed in vacuo on a rotavap.The residue was stirred with 500 mL ice water for fifteen minutes, thenextracted twice with 500 mL aliquots of ethyl acetate. The organicphases were combined and washed successively with cold 250 mL, aliquotsof saturated sodium chloride solution (two times), 1 N sodium hydroxidesolution, and again with saturated sodium chloride. The organic phasewas then dried over sodium sulfate, filtered and taken to dryness invacuo to give a yellowish white foam, which pulverized to a glass whentouched with a spatula. The powder that formed, 14.65 g analyzed as amixture of 82.1% 6 7.4% 8 and 5.7% 9 (by HPLC area %).

EXAMPLE 25 Scheme 1

Step 3C

Method B:

Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

A 5-L four neck flask was equipped as in the above example and 228.26 gacetic acid and 41.37 g sodium acetate were added with stirring undernitrogen. Using an oil bath, the mixture was heated to an internaltemperature of 100° C. The mesylate (123.65 g) was added, and heatingwas continued for thirty minutes. At the end of this period, heating wasstopped and 200 mL of ice water was added. The temperature dropped to40° C. and stirring was continued for 1 hour, after which the reactionmixture was poured slowly into 1.5 L of cold water in a 5-L stirredflask. The product separated as a gummy oil. The oil was dissolved in 1L ethyl acetate and washed with 1 L each cold saturated sodium chloridesolution, 1 N sodium hydroxide, and finally saturated sodium chlorideagain. The organic phase was dried over sodium sulfate and filtered. Thefiltrate was taken to dryness in vacuo to give a foam which collapsed toa gummy oil. This was triturated with ether for some time and eventuallysolidified. The solid was filtered and washed with more ether to afford79.59 g of a yellow white solid. This consisted of 70.4% of the desiredΔ⁹,11 enester 6, 12.3% of the Δ¹¹,12 enester 8, 10.8% of the7-α,9-α-lactone 9 and 5.7% unreacted 5.

EXAMPLE 26 Scheme 1

Step 3D

Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

A 4-neck jacketed 500 mL reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with 8.32 g of the crude enester in 83 mLmethylene chloride, with stirring under nitrogen. To this was added 4.02g dibasic potassium phosphate, followed by 12 mL oftrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 8° C. To theaddition funnel 36 mL of 30% hydrogen peroxide was added over a 10minute period. The initially pale yellow colored reaction mixture turnedalmost colorless after the addition was complete. The reaction mixtureremained at 9±1° C. throughout the addition and on continued stirringovernight (23 hours total) Methylene chloride (150 mL) was added to thereaction mixture and the entire contents were added to ˜250 mL icewater. This was extracted three times with 150 mL aliquots of methylenechloride. The combined methylene chloride extracts were washed with 400mL cold 3% sodium sulfite solution to decompose any residual peroxide.This was followed by a 330 mL cold 1 N sodium hydroxide wash, a 400 mLcold 1 N hydrochloric acid wash, and finally a wash with 400 mL brine.The organic phase was dried over magnesium sulfate, filtered, and thefilter cake was washed with 80 mL methylene chloride. Solvent wasremoved in vacuo to give 9.10 g crude product as a pale yellow solid.This was recrystallized from ˜25 mL 2-butanone to give 5.52 g nearlywhite crystals. A final recrystallization from acetone (˜50 mL gave 3.16g long, acicular crystals, mp 241-243° C.

EXAMPLE 27 Scheme 1

Step 3

Option 1:

From4'S(4α),7α-Hexadecahydro-11'α-hydroxy-10'β,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'β(2'H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

Diketone (20 g) was charged into a clean and dried reactor followed bythe addition of 820 ml of MeOH and 17.6 ml of 25% NaOMe/MeOH solution.The reaction mixture was heated to reflux condition (˜67° C.) for 16-20hours. The product was quenched with 40 mL of 4N HCl. The solvent wasremoved at atmospheric pressure by distillation. 100 mL of toluene wasadded and the residual methanol was removed by azeotrope distillationwith toluene. After concentration, the crude hydroxyester 4 wasdissolved in 206 mL of methylene chloride and cooled to 0° C.Methanesulfonyl chloride (5 mL) was added followed by a slow addition of10.8 ml of triethylamine. The product was stirred for 45 minutes. Thesolvent was removed by vacuum distillation to give the crude mesylate 5.

In a separate dried reactor was added 5.93 g of potassium formate, 240mL of formic acid and followed by 118 mL of acetic anhydride. Themixture was heated to 70° C. for 4 hours.

The formic acid mixture was added to the concentrated mesylate solution5 prepared above. The mixture was heated to 95-105° C. for 2 hours. Theproduct mixture was cooled to 50° C. and the volatile components wereremoved by vacuum distillations at 50° C. The product was partitionedbetween 275 ml of ethyl acetate and 275 ml of water. The aqueous layerwas back extracted with 137 ml of ethyl acetate, washed with 240 ml ofcold 1N sodium hydroxide solution and then 120 ml of saturated NaCl.After phase separation, the organic layer was concentrated to undervacuum distillation to give crude enester.

The product was dissolved in 180 mL of methylene chloride and cooled to0 to 15° C. 8.68 g of dipotassium hydrogen phosphate was added followedby 2.9 mL of trichloroacetonitrile. A 78 mL solution of 30% hydrogenperoxide was added to the mixture over a 3 minute period. The reactionmixture was stirred at 0-15° C. for 6-24 hours. After the reaction, thetwo phase mixture was separated. The organic layer was washed with 126mL of 3% sodium sulfite solution, 126 mL of 0.5 N sodium hydroxidesolution, 126 mL of 1 N hydrochloric acid and 126 mL of 10% brine. Theproduct was dried over anhydrous magnesium sulfate or filtered overCelite and the solvent methylene chloride was removed by distillation atatmospheric pressure. The product was crystallized from methylethylketone twice to give 7.2 g of eplerenone. ##STR125##

EXAMPLE 28 Scheme 1

Step 3

Option 2:

Conversion o1'S(4'α),7'α-Hexadecahydro-11'α-hydroxy-10'β,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'β(2'H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactonewithout intermediate.

A 4-neck 5-L round bottom flask was equipped with mechanical stirrer,addition funnel with nitrogen inlet tube, thermometer and condenser withbubbler attached to a sodium hypochlorite scrubber. The diketone (83.20g) was added to the flask in 3.05 L methanol. The addition funnel wascharged with 67.85 g of a 25% (w:w) solution of sodium methoxide inmethanol With stirring under nitrogen, the methoxide was added dropwiseto the flask over a 15 minute period. A dark orange/yellow slurrydeveloped. The reaction mixture was heated to reflux for 20 hours and175 mL 4 N hydrochloric acid was added dropwise while refluxingcontinued. (caution, HCN evolution during this operation!) The refluxcondenser was replaced with a takeoff head and 1.6 L of methanol wasremoved by distillation while 1.6 L of aqueous 10% sodium chloridesolution was added dropwise through the funnel, at a rate to match thedistillation rate. The reaction mixture was cooled to ambienttemperature and extracted twice with 2.25 L of aliquots of methylenechloride. The combined extracts were washed with cold 750 mL aliquots of1 N sodium hydroxide and saturated sodium chloride solution. The organiclayer was dried by azeotropic distillation of the methanol at oneatmosphere, to a final volume of 1 L (0.5% of the total was removed foranalysis).

The concentrated organic solution (hydroxyester) was added back to theoriginal reaction flask equipped as before, but without the HCN trap.The flask was cooled to 0° C. and 30.7 g methanesulfonyl chloride wasadded with stirring under nitrogen. The addition funnel was charged with32.65 g triethylamine, which was added dropwise over a 15 minute period,keeping 0the temperature at 5° C. Stirring was continued for 2 hours,while the reaction mixture warmed to ambient. A column consisting of 250g Dowex 50 W×8-100 acid ion exchange resin was prepared and was washedbefore using with 250 mL water, 250 mL methanol and 500 mL methylenechloride. The reaction mixture was run down this column and collected. Afresh column was prepared and the above process was repeated. A third250 g column, consisting of Dowex 1×8-200 basic ion exchange resin wasprepared and pretreated as in the acid resin treatment described above.The reaction mixture was run down this column and collected A fourthcolumn of the basic resin was prepared and the reaction mixture againwas run down the column and collected. Each column pass was followed bytwo 250 mL methylene chloride washes down the column, and each passrequired ˜10 minutes. The solvent washes were combined with the reactionmixture and the volume was reduced in vacuo to ˜500 mL and 2% of thiswas removed for qc. The remainder was further reduced to a final volumeof 150 ML (crude mesylate solution).

To the original 5-L reaction set-up was added 960 mL formic acid, 472 mLacetic anhydride and 23.70 g potassium formate. This mixture was heatedwith stirring under nitrogen to 70° C. for 16 hours. The temperature wasthen increased to 100° C. and the crude mesylate solution was added overa thirty minute period via the addition funnel. The temperature droppedto 85° C. as methylene chloride was distilling out of the reactionmixture. After all of it had been removed, the temperature climbed backto 100° C., and was held there for 2.5 hours. The reaction mixture wascooled to 40° C. and the formic acid was removed under pressure untilthe minimum stir volume had been reached (˜150 mL). The residue wascooled to ambient and 375 mL methylene chloride was added. The dilutedresidue was washed with cold 1 L portions of saturated sodium chloridesolution, 1 N sodium carbonate, and again with sodium chloride solution.The organic phase was dried over magnesium sulfate (150 g), and filteredto give a dark reddish brown solution (crude enester solution).

A 4-neck jacketed 1 L reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with the crude enester solution (estimated60 g) in 600 mL methylene chloride, with stirring under nitrogen. Tothis was added 24.0 g dibasic potassium phosphate, followed by 87 mLtrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 10° C. To theaddition funnel 147 mL 30% hydrogen peroxide was added mixture over a 30minute period. The initially dark reddish brown colored reaction mixtureturned a pale yellow after the addition was complete. The reactionmixture remained at 10±1° C. throughout the addition and on continuedstirring overnight (23 hours total). The phases were separated and theaqueous portion was extracted twice with 120 mL portions of methylenechloride. The combined organic phases were then washed with 210 mL 3%sodium sulfite solution was added. This was repeated a second time,after which both the organic and aqueous parts were negative forperoxide by starch/iodide test paper. The organic phase was successivelywashed with 210 mL aliquots of cold 1 N sodium hydroxide, 1 Nhydrochloric acid, and finally two washes with brine. The organic phasewas dried azeotropically to a volume of ˜100 mL, fresh solvent was added(250 mL and distilled azeotropically to the same 100 mL and theremaining solvent was removed in vacuo to give 57.05 g crude product asa gummy yellow foam. A portion (51.01 g) was further dried to a constantweight of 44.3 g and quantitatively analyzed by HPLC. It assayed at27.1% EPX.

EXAMPLE 29

11α-Hydroxyandrostendione (429.5 g) and toluene sulfonic acid hydrate(7.1) were charged to a reaction flask under nitrogen. Ethanol (2.58 L)was added to the reactor, and the resulting solution cooled to 50° C.Triethyl orthoformate (334.5 g) was added to the solution over a 15minute period at 0° to 15° C. After the triethyl orthoformate additionwas complete the reaction mixture was warmed to 40° C. and reacted atthat temperature for 2 hours, after which the temperature was increasedto reflux and reaction continued under reflux for an additional 3 hours.The reaction mixture was cooled under vacuum and the solvent removedunder vacuum to yield 3-ethoxyandrosta-3,5-diene-17-one.

EXAMPLE 30

Formation of Enamine from 11α-hydroxycanrenone ##STR126##

Sodium cyanide (1.72 g) was placed in 25 mL 3-neck flask fitted with amechanical stirrer Water (2.1 mL) was added and the mixture was stirredwith heating until the solids dissolved. Dimethylformamide (15 mL) wasadded followed by 11α-hydroxycanrenone (5.0 g). A mixture of water (0.4mL) and sulfuric acid (1.49 g) was added to mixture. The mixture washeated to 85° C. for 2.5 hours at which time HPLC analysis showedcomplete conversion to product. The reaction mixture was cooled to roomtemperature. Sulfuric acid (0.83 g) was added and the mixture stirredfor one half hour. The reaction mixture was added to 60 mL water cooledin an ice bath. The flask was washed with 3 mL DMF and 5 mL water. Theslurry was stirred for 40 min. and filtered The filter cake was washedtwice with 40 mL water and dried in a vacuum oven at 60° C. overnight toyield the 11α-hydroxy enamine, i.e.,5'R(5'α),7'β-20'-aminohexadecahydro-11'β-hydroxy-10'.alpha.,13'α-dimethyl-3',5-dioxospiro[furan-2(3H),17'α(5'H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile(4.9 g).

EXAMPLE 31

Conversion of 11α-hydroxycanrenone to Diketone ##STR127##

Sodium cyanide (1.03 g) was added to a 50 mL 3-neck flask fitted with amechanical stirrer. Water (1.26 mL) was added and the flask was heatedslightly to dissolve the solid. Dimethylacetamide [or dimethyformamide](9 mL) was added followed by 11α-hydroxycanrenone (3.0 g). A mixture ofsulfuric acid (0.47 mL) and water (0.25 mL) was added to the reactionflask while stirring. The mixture was heated to 95° C. for 2 hours. HPLCanalysis indicated that the reaction was complete. Sulfuric acid (0.27mL) was added and the mixture stirred for 30 min. Additional water (25mL) and sulfuric acid (0.90 mL) were introduced and the reaction mixturestirred for 16 hours. The mixture was then cooled in an ice bath to5-10° C. The solid was isolated by filtering through a sintered glassfilter followed by washing twice with water (20 mL). The solid diketone,i.e.,4'S(4'α),7'α-Hexadecahydro-11'α-hydroxy-10'β,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'β(2'H)-carbonitrilewas dried in a vacuum oven to yield 3.0 g of a solid.

EXAMPLE 32

A suspension of 5.0 g of the diketone produced in the manner describedin Example 31 in methanol (100 mL) was heated to reflux and a 25%solution of potassium methoxide in methanol (5.8 mL) was added over 1min. The mixture became homogeneous. After 15 min., a precipitate waspresent. The mixture was heated at reflux and again became homogeneousafter about 4 hours. Heating at reflux was continued for a total of 23.5hours and 4.0 N HCl (10 mL) was added. A total of 60 mL of a solution ofhydrogen cyanide in methanol was removed by distillation. Water (57 mL)as added to the distillation residue over 15 min. The temperature of thesolution was raised to 81.5° during water addition and an additional 4mL of hydrogen cyanide/methanol solution was removed by distillation.After water addition was complete, the mixture became cloudy and theheat source was removed. The mixture was stirred for 3.5 hours andproduct slowly crystallized. The suspension was filtered and thecollected solid was washed with water, dried in a stream of air on thefunnel, and dried at 92° (26 in. Hg) for 16 hours to give 2.98 g of anoff-white solid. The solid was 91.4% of the hydroxyester, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone by weight. The yield was 56.1%.

EXAMPLE 33

Diketone prepared in the manner described in Example 31 was charged intoa cleaned and dried 3-neck reaction flask equipped with a thermometer, aDean Stark trap and a mechanical stirrer Methanol (24 mL) was charged tothe reactor at room temperature (22° C.) and the resulting slurrystirred for 5 min. A 25% by weight solution of sodium methoxide inmethanol (52.8 mL) was charged to the reactor and the mixture stirredfor 10 min. at room temperature during which the reaction mixture turnedto a light brown clear solution and a slight exotherm was observed (2-3°C.). The addition rate was controlled to prevent the pot temperaturefrom exceeding 30° C. The mixture was thereafter heated to refluxconditions (about 67° C.) and continued under reflux for 16 hrs. Asample was then taken and analyzed by HPLC for conversion. The reactionwas continued under reflux until the residual diketone was not greaterthan 3% of the diketone charge. During reflux 4 N HCl (120 mL) wascharged to the reaction pot resulting in the generation of HCN which wasquenched in a scrubber.

After conclusion of the reaction, 90-95% of the methanol solvent wasdistilled out of the reaction mixture at atmospheric pressure. Headtemperature during distillation varied from 67-75° C. and the distillatewhich contained HCN was treated with caustic and bleach before disposal.After removal of methanol the reaction mixture was cooled to roomtemperature, solid product beginning to precipitate as the mixturecooled in the 40-45° C. range. An aqueous solution containing optionally5% by weight sodium bicarbonate (1200 mL) at 25° C. was charged to thecooled slurry and the resultant mixture then cooled to 0° C. in about 1hr. Sodium bicarbonate treatment was effective to eliminate residualunreacted diketone from the reaction mixture. The slurry was stirred at0° C. for 2 hrs. to complete the precipitation and crystallization afterwhich the solid product was recovered by filtration and the filter cakewashed with water (100 mL). The product was dried at 80-90° C. under 26"mercury vacuum to constant weight. Water content after drying was lessthan 0.25% by weight. Adjusted molar yield was around 77-80% by weight.

EXAMPLE 34

Diketone as prepared in accordance with Example 31 (1 eq.) was reactedwith sodium methoxide (4.8 eqs.) in a methanol solvent in the presenceof zinc iodide (1 eq.). Work up of the reaction product can be either inaccordance with the extractive process described herein, or by anon-extractive process in which methylene chloride extractions, brineand caustic washes, and sodium sulfate drying steps are eliminated. Alsoin the non-extractive process, toluene was replaced with 5% by weightsodium bicarbonate solution.

EXAMPLE 35

The hydroxyester prepared as by Example 34 (1.97 g) was combined withtetrahydrofuran (20 mL) and the resulting mixture cooled to -70° C.Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30min., after which imidazole (1.3 g) was added. The reaction mixture waswarmed to room temperature and stirred for an additional 2 hrs. Themixture was then diluted with methylene chloride and extracted withwater. The organic layer was concentrated to yield crude enester (1.97g). A small sample of the crude product was analyzed by HPLC. Theanalysis showed that the ratio of 9,11-olefin:11,12-olefin:7,9-lactonewas 75.5:7.2:17.3. When carried out at 0° C. but otherwise as describedabove, the reaction yielded a product in which the9,11-olefin:11,12-olefin:7,9-lactone distribution was 77.6:6.7:15.7.This procedure combines into one step the introduction of a leavinggroup and elimination thereof for the introduction of the 9,11-olefinstructure of the enester, i.e., reaction was sulfuryl chloride causesthe 11α-hydroxy group of the hydroxy ester of Formula V to be replacedby halide and this is followed by dehydrohalogenation to the Δ-9,11structure. Thus formation of the enester is effected without the use ofa strong acid (such as formic) or a drying agent such as aceticanhydride. Also eliminated is the refluxing step of the alternativeprocess which generates carbon monoxide.

EXAMPLE 36

Hydroxyester (20 g) prepared as by Example 34, and methylene chloride(400 mL) were added to a clean dry three-neck round bottom flask fittedwith a mechanical stirrer, addition funnel and thermocouple. Theresulting mixture was stirred at ambient temperature until completesolution was obtained. The solution was cooled to 5° C. using an icebath. Methanesulfonyl chloride (5 mL) was added to the solution of CH₂Cl₂ containing the hydroxyester, rapidly followed by the slow dropwiseaddition of triethylamine (10.8 mL). The addition rate was adjusted sothat the temperature of the reaction did not exceed 5° C. The reactionwas very exothermic; therefore cooling was necessary. The reactionmixture was stirred at about 5° C. for 1 h. When the reaction wascomplete (HPLC and TLC analysis), the mixture was concentrated at about0° C. under 26 in Hg vacuum until it became a thick slurry. Theresulting slurry was diluted with CH₂ Cl₂ (160 mL), and the mixture wasconcentrated at about 0° C. under 26 in Hg vacuum to obtain aconcentrate The purity of the concentrate (mesylate product of FormulaIV wherein R³ =H and --A--A-- and --B--B-- are both --CH₂ --CH₂ --,i.e., methyl hydrogen11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone tomethyl hydrogen17α-hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone was found to be 82% (HPLC area %). This material was used forthe next reaction without isolation.

Potassium formate (4.7 g), formic acid (16 mL) and acetic anhydride (8mL, 0.084 mol) were added to a clean dry reactor equipped withmechanical stirrer, condenser, thermocouple and heating mantle. Theresulting solution was heated to 70° C. and stirred for about 4-8 hoursThe addition of acetic anhydride is exothermic and generated gas (CO),so that the rate of addition had to be adjusted to control bothtemperature and gas generation (pressure). The reaction time to preparethe active eliminating reagent was dependent on the amount of waterpresent in the reaction (formic acid and potassium formate containedabout 3-5% water each). The elimination reaction is sensitive to theamount of water present; if there is >0.1% water (KF), the level of the7,9-lactone impurity may be increased. This by product is difficult toremove from the final product. When the KF showed <0.1% water, theactive eliminating agent was transferred to the concentrate of mesylate(0.070 mol) prepared in the previous step. The resulting solution washeated to 95° C. and the volatile material was distilled off andcollected in a Dean Stark trap. When volatile material evolution ceased,the Dean Stark trap was replaced with the condenser and the reactionmixture was heated for additional 1 h at 95° C. Upon completion (TLC andHPLC analysis; <0.1% starting material) the content was cooled to 50° C.and vacuum distillation was started (26 in Hg/50° C.). The mixture wasconcentrated to a thick slurry and then cooled to ambient temperature.The resulting slurry was diluted with ethyl acetate (137 mL) and thesolution was stirred for 15 min. and diluted with water (137 mL). Thelayers were separated, and the aqueous lower layer was re-extracted withethyl acetate (70 mL). The combined ethyl acetate solution was washedonce with brine solution (120 mL) and twice with ice cold IN NaOHsolution (120 mL each). The pH of aqueous was measured, and the organiclayer rewashed if the pH of the spent wash liquor was <8. When the pH ofthe spent wash was observed to be >8, the ethyl acetate layer was washedonce with brine solution (120 mL) and concentrated to dryness by rotaryevaporation using a 50° C. water bath. The resulting enester, solidproduct i.e., methyl hydrogen17α-hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-lactoneweighed 92 g (77% mol yield).

EXAMPLE 37

Hydroxyester (100 g; 0.22 mol) prepared as by Example 34 was charged toa 2 L 3-neck round bottom flask equipped with mechanical stirrer,addition funnel, and thermocouple. A circulating cooling bath was usedwith automatic temperature control. The flask was dried prior toreaction because of the sensitivity of methanesulfonyl chloride towater.

Methylene chloride (1 L) was charged to the flask and the hydroxyesterdissolved therein under agitation. The solution was cooled to 0° C. andmethane sulfonyl chloride (25 mL; 0.32 mol) was charged to the flask viathe addition funnel. Triethylamine (50 mL; 0.59 mol) was charged to thereactor via the addition funnel and the funnel was rinsed withadditional methylene chloride (34 mL). Addition of triethylamine washighly exothermic. Addition time was around 10 min. under agitation andcooling. The charge mixture was cooled to 0° C. and held at thattemperature under agitation for an additional 45 min. during which thehead space of the reaction flask was flushed with nitrogen. A sample ofthe reaction mixture was then analyzed by thin layer chromatography andhigh performance liquid chromatography to check for reaction completion.The mixture was thereafter stirred at 0° C. for an additional 30 min.and checked again for reaction completion. Analysis showed the reactionto be substantially complete at this point; the solvent methylenechloride was stripped at 0° C. under 26" mercury vacuum. Gaschromatography analysis of the distillate indicated the presence of bothmethane sulfonyl chloride and triethylamine. Methylene chloride (800 mL)was thereafter charged to the reactor and the resulting mixture wasstirred for 5 min. at a temperature in the range of 0-15° C. The solventwas again stripped at 0-5° C. under 26" mercury vacuum yielding themesylate of Formula IV wherein R³ is H, --A--A-- and --B--B-- are --CH₂--CH₂ -- and R¹ is methoxy carbonyl. The purity of the product was about90-95 area %.

To prepare an elimination reagent, potassium formate (23.5 g; 0.28 mol),formic acid (80 mL) and acetic anhydride (40 mL) were mixed in aseparate dried reactor. Formic acid and acetic anhydride were pumpedinto the reactor and the temperature was maintained not greater than 40°C. during addition of acetic anhydride. The elimination reagent mixturewas heated to 70° C. to scavenge water from the reaction system. Thisreaction was continued until the water content was lower than 0.3% byweight as measured by Karl Fisher analysis. The elimination reagentsolution was then transferred to the reactor containing the concentratedcrude mesylate solution prepared as described above. The resultingmixture was heated to a maximum temperature of 95° C. and volatiledistillate collected until no further distillate was generated.Distillation ceased at about 90° C. After distillation was complete, thereaction mixture was stirred at 95° C. for an additional 2 hrs. andcompletion of the reaction was checked for thin layer chromatography.When the reaction was complete, the reactor was cooled to 50° C. and theformic acid and solvent removed from the reaction mixture under 26"mercury vacuum at 50° C. The concentrate was cooled to room temperatureand thereafter ethyl acetate (688 mL) was introduced and the mixture ofethyl acetate and concentrate stirred for 15 min. At this point, a 12%brine solution (688 mL) was introduced to assist in removing watersoluble impurities from the organic phase. The phases were then allowedto settle for 20 min. The aqueous layer was transferred to anothervessel to which an additional amount of ethyl acetate (350 mL) wascharged. This back extraction of the aqueous layer was carried out for30 min. after which the phases were allowed to settle and the ethylacetate layers combined. To the combined ethyl acetate layers, saturatedsodium chloride solution (600 mL) was charged and stirring carried outfor 30 min. The phases were then allowed to settle. The aqueous layerwas removed. An additional sodium chloride (600 mL) wash was carriedout. The organic phase was separated from the second spent wash liquor.The organic phase was then washed with 1 N sodium hydroxide (600 mL)under stirring for 30 min. The phases were settled for 30 min. to removethe aqueous layer. The pH of the aqueous layer was checked and it foundto be >7. A further wash was carried out with saturated sodium chloride(600 mL) for 15 min. The organic phase was finally concentrated under26" mercury vacuum at 50° C. and the product recovered by filtration.The final product was a foamy brown solid when dried. Further drying at45° C. under reduced pressure for 24 hrs. yielded 95.4 g of the enesterproduct which assayed at 68.8%. The molar yield was 74.4% corrected forboth the starting hydroxy ester and the final enester.

EXAMPLE 38

The procedure of Example 37 was repeated except that the multiplewashing steps were avoided by treating the reaction solution with an ionexchange resin. Basic alumina or basic silica. Conditions for treatmentwith basic silica are set forth in Table 38. Each of these treatmentswas found effective for removal of impurities without the multiplewashes of Example 44.

                  TABLE 38                                                        ______________________________________                                        Factor Set point                                                                              Purpose of Experiment                                                                           Key results                                 ______________________________________                                        Basic  2 g/125 g                                                                              Treating the reaction mixture                                                                   The yield                                     alumina product with basic alumina to remove was 93%                            Et.sub.3 N.HCl salt and to                                                    eliminate the 1N NaOH and 1N                                                  HCl washes                                                                  Basic 2 g/125 g Treating the reaction mixture The yield                       silica product with basic silica which is was 95%                               cheaper to remove Et.sub.3 N.HCl                                              salt and eliminate 1N NaOH                                                    and 1N HCl washes                                                         ______________________________________                                    

EXAMPLE 39

Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL) were mixed ina 100 mL reactor. trifluoroacetic anhydride (9.5 mL) was added to themixture at a rate controlled to maintain temperature during additionbelow 30° C. The solution was then heated to 30° C. for 30 min. toprovide an elimination reagent useful for converting the mesylate ofFormula IV to the enester of Formula II.

The preformed TFA/TFA anhydride elimination reagent was added to apreviously prepared solution of the mesylate of Formula IV. Theresulting mixture was heated at 40° C. for 41/2 hrs., the degree ofconversion being periodically checked by TLC or HPLC. When the reactionwas complete, the mixture was transferred to 1-neck flask andconcentrated to dryness under reduced pressure at room temperature (22°C.). Ethyl acetate (137 mL) was added to the mixture to obtain completedissolution of solid phase material after which a water/brine mixture(137 mL) was added and the resulting two phase mixture stirred for 10min. The phases were then allowed to separate for 20 min. Brine strengthwas 24% by weight. The aqueous phase was contacted with an additionalamount of ethyl acetate (68 mL) and the two phase mixture thus preparedwas stirred for 10 min. after which it was allowed to stand for 15 min.for phase separation. The ethyl acetate layers from the two extractionswere combined and washed with 24% by weight brine (120 mL), anotheraliquot of 24% by weight brine (60 mL), 1 N sodium hydroxide solution(150 mL) and another portion of brine (60 mL). After each aqueous phaseaddition, the mixture was stirred for 10 min. and allowed to stand for15 min. for separation. The resulting solution was concentrated todryness under reduced pressure at 45° C. using a water aspirator. Thesolid product (8.09 g) was analyzed by HPLC and found to include 83.4area % of the enester, 2.45 area % of the 11,12-olefin, 1.5% of the7,9-lactone, and 1.1% of unreacted mesylate.

EXAMPLE 40

The mesylate having the structure prepared per Example 23 (1.0 g),isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) were placedin a 50 ml flask and heated to 9° C. with stirring. After 5 hours themixture was cooled to 25° C. and concentrated in vacuo at 10 mm of Hg.The residue was dissolved in CH₂ Cl₂ (20 ml) and washed with 5% aqueousNaHCO₃. The CH₂ Cl₂ layer was concentrated in vacuo to give 1.47 g of atan oil. This material was recrystallized from CH₂ Cl₂ /Et₂ O to give0.50 g of enol acetate of Formula IV(Z).

This material was added to a mixture of sodium acetate (0.12 g) andacetic acid (2.0 ml) that had been previously heated to 100° C. withstirring. After 60 minutes the mixture was cooled to 25° C. and dilutedwith CH₂ Cl₂ (20 ml). The solution was washed with water (20 ml) anddried over MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated in vacuo to give 0.4 g of the desired9,11-olefin, IV(Y). The crude product contained less than 2% of the7,9-lactone impurity.

EXAMPLE 41

Thermo Elimination of Mesylate in DMSO ##STR128##

A mixture of 2 g of mesylate and 5 ml of DMSO in a flask was heated at80° C. for 22.4 hours. HPLC analysis of the reaction mixture indicatedno starting material was detected. To the reaction was added water (10ml) and the precipitate was extracted with methylene chloride threetimes. The combined methylene chloride layers were washed with water,dried over magnesium sulfate, and concentrated to give the enester.

EXAMPLE 42

In a 50 mL pear-shaped flask under stirring the enester of Formula IIA(1.07 g assaying 74.4% enester), trichioroacetamide (0.32 g),dipotassium hydrogen phosphate (0.70 g) as solid were mixed withmethylene chloride (15.0 mL). A clear solution was obtained. Hydrogenperoxide (30% by weight; 5.0 mL) was added via a pipet over a 1 min.period. The resulting mixture was stirred for 6 hrs. at room temperatureat which point HPLC analysis showed that the ratio of epoxymexrenone toenester in the reaction mixture was approximately 1:1. Additionaltrichloroacetamide (0.32 g) was added to the reaction mixture andreaction continued under agitation for 8 more hours after which time theremaining proportion of enester was shown to have been reduced to 10%.Additional trichioroacetamide (0.08 g) was added and the reactionmixture was allowed to stand overnight at which point only 5% ofunreacted enester remained relative to epoxymexrenone in the mixture.

EXAMPLE 43

Enester of Formula IIA (5.4 g, assaying 74.4% enester) was added to a100 mL reactor. Trichloroacetamide (4.9 g) and dipotassium hydrogenphosphate (3.5 g) both in solid form were added to the enester followedby methylene chloride (50 mL). The mixture was cooled to 15° C. and a30% hydrogen peroxide (25 g) was added over a ten min. period. Thereaction mixture was allowed to come to 20° C. and stirred at thattemperature for 6 hrs., at which point conversion was checked by HPLC.Remaining enester was determined to be less than 1% by weight.

The reaction mixture was added to water (100 mL), the phases wereallowed to separate, and the methylene chloride layer was removed.Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloridelayer. After 20 min. the phases were allowed to separate HCl (0.5 N; 50mL) was added to the methylene chloride layer after which the phaseswere allowed to separate and the organic phase was washed with saturatedbrine (50 mL). The methylene chloride layer was dried over anhydrousmagnesium sulfate and the solvent removed. A white solid (5.7 g) wasobtained. The aqueous sodium hydroxide layer was acidified and extractedand the extract worked up to yield an additional 0.2 g of product. Yieldof epoxymexrenone was 90.2%.

EXAMPLE 44

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 with the following differences: the initialcharge comprised of enester (5.4 g assaying 74.4% enester),trichloroacetamide (3.3 g), and dipotassium hydrogen phosphate (3.5 g).Hydrogen peroxide solution (12.5 mL) was added. The reaction wasconducted overnight at 20° C. after which HPLC showed a 90% conversionof enester to epoxymexrenone. Additional trichloroacetamide (3.3 g) and30% hydrogen peroxide (5.0 mL) was added and the reaction carried outfor an additional 6 hrs. at which point the residual enester was only 2%based on the enester charge. After work up as described in Example 43,5.71 g of epoxymexrenone resulted.

EXAMPLE 45

The enester of Formula IIA was converted to epoxymexrenone in the mannergenerally described in Example 43. In the reaction of this Example,enester charge was 5.4 g (assaying 74.4% enester), thetrichloroacetamide charge was 4.9 g, hydrogen peroxide charge was 25 g,dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at20° C. for 18 hrs. The residual enester was less than 2%. After work up,5.71 g of epoxymexrenone resulted.

EXAMPLE 46

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 except that the reaction temperature in thisExample was 28° C. The materials charged in the reactor included enester(2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7g), hydrogen peroxide (17.0 g) and methylene chloride (50 mL). After 4hrs. reaction, unreacted enester was only 2% based on the enestercharge. After work up as described in Example 43, 3.0 g ofepoxymexrenone was obtained.

EXAMPLE 47

Enester of Formula IIA (17 g assaying 72% enester) was dissolved inmethylene chloride (150 mL) after which trichloroacetamide (14.9 g) wasadded under slow agitation. The temperature of the mixture was adjustedto 25° C. and the solution of dipotassium hydrogen phosphate (10.6 g) inwater (10.6 mL) was stirred into the enester substrate solution under400 rpm agitation. Hydrogen peroxide (30% by weight solution; 69.4 mL)was added to the substrate/phosphate/trichloroacetamide solution over a3-5 min. period. No exotherm or oxygen evolution was observed. Thereaction mixture thus prepared was stirred at 400 rpm and 25° C. for18.5 hrs. No oxygen evolution was observed throughout the course of thereaction. The reaction mixture was diluted with water (69.4 mL) and themixture stirred at about 250 rpm for 15 min. No temperature control wasnecessary for this operation and it was conducted essentially at roomtemperature (any temperature in the range of 5-25° C. being acceptable).The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer was removed.

The aqueous layer was back extracted with methylene chloride (69.4 mL)for 15 min. under agitation of 250 rpm. The layers were allowed toseparate and the lower methylene chloride layer was removed. The aqueouslayer (177 g; pH=7) was submitted for hydrogen peroxide determination.The result (12.2%) indicating that only 0.0434 mol of hydrogen peroxidewere consumed in the reaction was 0.0307 mol of olefin. Back extractionwith a small amount of methylene chloride volume was sufficient toinsure no loss of epoxymexrenone in the aqueous layer. This result wasconfirmed with the application of a second large methylene chlorideextraction in which only trichloroacetamide was recovered.

The combined methylene chloride solutions from the above describedextractions were combined and washed with 3% by weight sodium sulfitesolution (122 mL) for at least 15 min. at about 250 rpm. A negativestarch iodide test (KI paper; no color observed; in a positive test apurple coloration indicates the presence of peroxide) was observed atthe end of the stir period.

The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer removed. The aqueous layer (pH=6) wasdiscarded. Note that addition of sodium sulfite solution can cause aslight exotherm so that such addition should be carried out undertemperature control.

The methylene chloride phase was washed with 0.5 N sodium hydroxide (61mL) for 45 min. at about 250 rpm and a temperature in the range of15-25° C. (pH=12-13). Impurities derived from trichloroacetamide wereremoved in this process. Acidification of the alkaline aqueous fractionfollowed by extraction of the methylene chloride confirmed that verylittle epoxymexrenone was lost in this operation.

The methylene chloride phase was washed once with 0.1 N hydrochloricacid (61 mL) for 15 min. under 250 rpm agitation at a temperature in therange 15-25° C. The layers were then allowed to separate and the lowermethylene chloride layer removed and washed again with 10% by weightaqueous sodium chloride (61 mL) for 15 min at 250 rpm at a temperaturein the range of 15-25° C. Again the layers were allowed to separate andthe organic layer removed. The organic layer was filtered through a padof Solkafloc and then evaporated to dryness under reduced pressure.Drying was completed with a water bath temperature of 65° C. Anoff-white solid (17.95 g) was obtained and submitted for HPLC assay.Epoxymexrenone assay was 66.05%. An adjusted molar yield for thereaction was 93.1%.

The product was dissolved in hot methyl ethyl ketone (189 mL) and theresulting solution was distilled at atmospheric pressure until 95 mL ofthe ketone solvent had been removed. The temperature was lowered to 50°C. as the product crystallized. Stirring was continued at 50° C. for 1hr. The temperature was then lowered to 20-25° C. and stirring continuedfor another 2 hrs. The solid was filtered and rinsed with MEK (24 mL)and the solid dried to a constant weight of 9.98 g, which by HPLC assaycontain 93.63% epoxymexrenone. This product was re-dissolved in hot MEK(106 mL) and the hot solution filtered through a 10 micron line filterunder pressure. Another 18 mL of MEK was applied as a rinse and thefiltered MEK solution distilled at atmospheric pressure until 53 mL ofsolvent had been removed. The temperature was lowered to 50° C. as theproduct crystallized; and stirring was continued at 50° C. for 1 hr. Thetemperature was then lowered to 20-25° C. and held at that temperaturewhile stirring was continued for another 2 hrs. The solid product wasfiltered and rinsed with MEK (18 mL). The solid product was dried to aconstant weight of 8.32 g which contained 99.6% epoxymexrenone perquantitative HPLC assay. The final loss on drying was less than 1.0%.Overall yield of epoxymexrenone in accordance with the reaction and workup of this Example is 65.8%. This overall yield reflected a reactionyield of 93%, an initial crystallization recovery of 78.9%, and arecrystallization recovery of 89.5%.

EXAMPLE 48

Epoxidation of Formula IIA Using Toluene

The enester of Formula IIA was converted to eplerenone in the methodgenerally described in Example 46 except that toulene was used as thesolvent. The materials charged to the reactor included enester (2.7 g)trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g),hydrogen peroxide (17.0 g) and toulene (50 ml). The reaction was allowedto exotherm to 28° C. and was complete in 4 hours. The resulting threephase mixture was cooled to 15° C., filtered, washed with water anddried in vacuo to yield 2.5 g of product.

EXAMPLE 49

Epoxidation of 9,11-Dienone

A compound designated XVIIA (compound XVII wherein --A--A-- and --B--B--are both --CH₂ --CH₂ --) (40.67 g) was dissolved in methylene chloride(250 mL) in a one liter 3 necked flask and cooled by ice salt mixtureexternally. Dipotassium phosphate (22.5 g), and trichloroacetonitrile(83.5 g) were added and mixture cooled to 2° C. after which 30% Hydrogenperoxide (200 g) was slowly added over a period of 1 hour. The reactionmixture was stirred at 120 for 8 hours and 14 hours at room temperature.A drop of the organic layer was taken and checked for any starting enoneand was found to be <0.5%. Water (400 mL) was added, stirred for 15 min.and layers separated. The organic layer was washed successively with 200mL of potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100mL of saturated sodium bicarbonate solution separating layers each time.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to yield crude epoxide (41 g). The product crystallizedfrom ethyl acetate:methylene chloride to give 14.9 g of pure material.

EXAMPLE 50

Epoxidation of Compound XVIIA Using m-chloroperbenzoic Acid

Compound XVIIA (18.0 g) was dissolved in 250 mL of methylene chlorideand cooled to 10° C. Under stirring solid m-chloroperbenzoic acid,(50-60% pure, 21.86 g) was added during 15 min. No rise in temperaturewas observed. The reaction mixture was stirred for 3 hours and checkedfor the presence of the dienone. The reaction mixture was treatedsuccessively with sodium sulfite solution (10%), sodium hydroxidesolution (0.5N), hydrochloric acid solution (5%) and finally with 50 mLof saturated brine solution. After drying with anhydrous magnesiumsulfate and evaporation, 17.64 g of the epoxide resulted and was useddirectly in the next step. The product was found to containBayer-Villager oxidation product that had to be removed by triturationfrom ethyl acetate followed by crystallization from methylene chloride.On a 500 g scale, the precipitated m-chlorobenzoic acid was filteredfollowed by the usual work up.

EXAMPLE 51

Epoxidation of Compound XVIIA Using Trichloroacetamide

Compound XVIIA (2 g) was dissolved in 25 mL of methylene chloride.Trichloroacetamide (2 g), dipotassium phosphate (2 g) were added. Understirring at room temperature 30% hydrogen peroxide (10 mL) was added andstirring continued for 18 hours to yield the epoxide (1.63 g).Bayer-Villager product was not formed.

EXAMPLE 52

Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 eq.) was charged to a2000 mL flask and slurried with dimethylsulfoxide (750.0 mL) at ambienttemperature. A trienone corresponding to Formula XX (wherein R³ is H and--A--A-- and --B--B-- are each --CH₂ --CH₂ --) (100.00 g; 335.01 mmol;1.00 eq.) was charged to the flask together with THF (956.0 mL).Trimethylsulfonium methylsulfate (126.14 g; 670.02 mmol; 2.00 eq.) wascharged to the flask and the resulting mixture heated at reflux, 80 to85° C. for 1 hr. Conversion to the 17-spirooxymethylene was checked byHPLC. THF approximately 1 L was stripped from the reaction mixture undervacuum after which water (460 mL) was charged over a 30 min. periodwhile the reaction mixture was cooled to 15° C. The resulting mixturewas filtered and the solid oxirane product washed twice with 200 mLaliquots of water. The product was observed to be highly crystalline andfiltration was readily carried out. The product was thereafter driedunder vacuum at 40° C. 104.6 g of the 3-methyl enol etherΔ-5,6,9,11,-17-oxirane steroid product was isolated.

EXAMPLE 53

Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) was charged to a dry500 mL reactor under a nitrogen blanket. Ethanol (270.9 mL) was chargedto the reactor and the sodium methoxide slurried in the ethanol. Diethylmalonate (103.90 g; 648.68 mmol; 2.00 eq.) was charged to the slurryafter which the oxirane steroid prepared in the manner described inExample 52 (104.60 g; 324.34 mmol; 1.00 eq.) was added and the resultingmixture heated to reflux, i.e., 80 to 85° C. Heating was continued for 4hrs. after which completion of the reaction was checked by HPLC. Water(337.86 mL) was charged to the reaction mixture over a 30 min. periodwhile the mixture was being cooled to 15° C. Stirring was continued for30 min. and then the reaction slurry filtered producing a filter cakecomprising a fine amorphous powder. The filter cake was washed twicewith water (200 mL each) and thereafter dried at ambient temperatureunder vacuum. 133.8 g of the 3-methylenolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonyl intermediate wasisolated.

EXAMPLE 54

The 3-methyl enolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonylintermediate (Formula XVIII where R³ is H and --A--A-- and --B--B-- areeach --CH₂ --CH₂ --; 133.80 g; 313.68 mmol; 1.00 eq., as produced inExample 53, was charged to the reactor together with sodium chloride(27.50 g; 470.52 mmol; 1.50 eq.) dimethyl formamide (709 mL) and water(5 mL) were charged to a 2000 mL reactor under agitation. The resultingmixture was heated to reflux, 138 to 142° C. for 3 hrs. after which thereaction mixture was checked for completion of the reaction by HPLC.Water was thereafter added to the mixture over a 30 min. period whilethe mixture was being cooled to 15° C. Agitation was continued for 30min. after which the reaction slurry was filtered recovering amorphoussolid reaction product as a filter cake. The filter cake was washedtwice (200 mL aliquots of water) after which it was dried. The product3-methylenolether-17-spirolactone was dried yielding 91.6 g (82.3%yield; 96 area % assay).

EXAMPLE 55

The enol ether produced in accordance with Example 54 (91.60 g; 258.36mmol; 1.00 eq.) ethanol (250 mL) acetic acid (250 mL) and water (250 mL)were charged to a 2000 mL reactor and the resulting slurry heated toreflux for 2 hrs. Water (600 mL) was charged over a 30 min. period whilethe reaction mixture was being cooled to 15° C. The reaction slurry wasthereafter filtered and the filter cake washed twice with water (200 mLaliquots). The filter cake was then dried; 84.4 g of product 3-ketoΔ4,5,9,11-17-spirolactone was isolated (compound of Formula XVII whereR³ is H and --A--A-- and --B--B-- are --CH₂ --CH₂ --; 95.9% yield)

EXAMPLE 56

Compound XVIIA (1 kg; 2.81 moles) was charged together with carbontetrachloride (3.2 L) to a 22 L 4-neck flask. N-bromo-succinamide (538g) was added to the mixture followed by acetonitrile (3.2 L). Theresulting mixture was heated to reflux and maintained at the 68° C.reflux temperature for approximately 3 hrs. producing a clear orangesolution. After 5 hrs. of heating, the solution turned dark. After 6hrs. the heat was removed and the reaction mixture was sampled. Thesolvent was stripped under vacuum and ethyl acetate (6 L) added to theresidue in the bottom of the still. The resultant mixture was stirredafter which a 5% sodium bicarbonate solution (4 L) was added and themixture stirred for 15 min. after which the phases were allowed tosettle. The aqueous layer was removed and saturated brine solution (4 L)introduced into the mixture which was then stirred for 15 min. Thephases were again separated and the organic layer stripped under vacuumproducing a thick slurry. Dimethylformamide (4 L) was then added andstripping continued to a pot temperature of 55° C. The still bottomswere allowed to stand overnight and DABCO (330 g) and lithium bromide(243 g) added. The mixture was then heated to 70° C. After one andone-half hrs. heating, a liquid chromatography sample was taken andafter 3.50 hrs. heating, additional DABCO (40 g) was added. After 4.5hrs. heating, water (4 L) was introduced and the resulting mixture wascooled to 15° C. The slurry was filtered and the cake washed with water(3 L) and dried on the filter overnight. The wet cake (978 g) wascharged back into the 22 L flask and dimethylformamide (7 L) added. Themixture thus produced was heated to 105° C. at which point the cake hadbeen entirely taken up into solution. The heat was removed and themixture in the flask was stirred and cooled. Ice water was applied tothe reactor jacket and the mixture within the reactor cooled to 14° C.and held for two hours. The resulting slurry was filtered and washedtwice with 2.5 L aliquots of water. The filter cake was dried undervacuum overnight. A light brown solid product 510 g was obtained.

EXAMPLE 57

To a 2 L 4-neck flask were charged: 9,11-epoxy canrenone as produced inExample 49, 50, or 51 (100.00 g; 282.1 mmol; 1.00 eq.),dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.7 mmol;2.51 eq.), and acetone cyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00eq.). The resulting suspension was mechanically stirred and treated withtetramethyl guanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 eq.). Thesystem was then filtered with a water cooled condenser and a dry icecondenser (filled with dry ice in acetone) to prevent escape of HCN. Thevent line from the dry ice condenser passed into a scrubber filled witha large excess of chlorine bleach. The mixture was heated to 80° C.

After 18 hrs., a dark reddish-brown solution was obtained which wascooled to room temperature with stirring. During the cooling process,nitrogen was sparged into the solution to remove residual HCN with thevent line being passed into bleach in the scrubber. After two hrs. thesolution was treated with acetic acid (72 g) and stirred for 30 min. Thecrude mixture was then poured into ice water (2 L) with stirring. Thestirred suspension was further treated with 10% aqueous HCl (400 mL) andstirred for 1 hr. Then the mixture was filtered to give a dark brick-redsolid (73 g). The filtrate was placed in a 4 L separatory funnel andextracted with methylene chloride (3×800 mL); and the organic layerswere combined and back extracted with water (2×2 L). The methylenechloride solution was concentrated in vacuo to give 61 g of a dark redoil.

After the aqueous wash fractions were allowed to sit overnight, aconsiderable precipitate developed. This precipitate was collected byfiltration and determined to be pure product enamine (14.8 g).

After drying the original red solid (73 g) was analyzed by HPLC and itwas determined that the major component was the 9,11-epoxyenamine. HPLCfurther showed that enamine was the major component of the red oilobtained from methylene chloride workup. Calculated molar yield ofenamine was 46%.

EXAMPLE 58

9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00 eq.) as prepared inaccordance with Example 57 was introduced into a 1000 mL round bottomflask. Methanol (300 mL) and 0.5% by weight aqueous HCl (192 mL) wereadded to the mixture which was thereafter refluxed for 17 hrs. Methanolwas thereafter removed under vacuum reducing the amount of material inthe still pot to 50 mL and causing a white precipitate to be formed.Water (100 mL) was added to the slurry which was thereafter filteredproducing a white solid cake which was washed three times with water.Yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).

EXAMPLE 59

The epoxydiketone prepared in accordance with Example 58 (200 mg; 0.49mmol) was suspended in methanol (3 mL) and1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) added to the mixture. Uponheating under reflux for 24 hrs. the mixture became homogeneous. It wasthen concentrated to dryness at 30° C. on a rotary evaporator and theresidue partitioned between methylene chloride and 3.0 N HCl.

Concentration of the organic phase yielded a yellow solid (193 mg) whichwas determined to be 22% by weight epoxy mexrenone. The yield was 20%.##STR129##

EXAMPLE 60

To 100 mg of the diketone suspended in 1.5 mL of methanol was added 10microliters (0.18 eq) of a 25% (w/w) solution of sodium methoxide inmethanol. The solution was heated to reflux. After 30 min. no diketoneremained and the 5-cyanoester was present. To the mixture was added 46microliters of 25% (w/w) sodium methanol solution in methanol. Themixture was heated at reflux for 23 hours at which time the majorproduct was eplerenone as judged by HPLC. ##STR130##

EXAMPLE 61

To 2 g of the diketone suspended in 30 ml of dry methanol was added 0.34mL of triethylamine. The suspension was heated at reflux for 4.5 hours.The mixture was stirred at 25° C. for 16 hours. The resulting suspensionwas filtered to give 1.3 g of the 5-cyanoester as a white solid.

To 6.6 g of the diketone suspended in 80 mL of methanol was added 2.8 mLof triethylamine. The mixture was heated at reflux for 4 hours and wasstirred at 25× for 88 hours during which time the product crystallizedfrom solution. Filtration followed by a methanol wash gave 5.8 g of thecyanoester as a white powder. The material was recrystallized fromchloroform/methanol to give 3.1 g of crystalline material which washomogeneous by HPLC.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above compositions and processeswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A process for the preparation of a compound ofFormula II: ##STR131## wherein --A--A-- represents the group --CHR⁴--CHR⁵ -- or --CR⁴ ═CR⁵ --R³, R⁴ and R⁵ are independently selected fromthe group consisting of hydrogen, halo, hydroxy, lower alkyl, loweralkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano, aryloxy, R¹represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical, --B--B-- represents the group --CHR⁶ --CHR⁷ -- or an alpha- orbeta-oriented group: ##STR132## where R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen, halo, lower alkoxy,acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, and R⁸ and R⁹ are independently selectedfrom the group consisting of hydrogen, halo, lower alkoxy, acyl,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, or R⁸ and R⁹ together comprise acarbocyclic or heterocyclic ring structure, or R⁸ or R⁹ together with R⁶or R⁷ comprise a carbocyclic or heterocyclic ring structure fused to thepentacyclic D ring;the process comprising: removing an 11α-leaving groupfrom a compound of Formula IV: ##STR133## wherein --A--A--, R¹, R³,--B--B--, R⁸, and R⁹ are as defined above, and R² is a leaving group theabstraction of which is effective for generating a double bond betweenthe 9- and 11-carbon atoms.
 2. A process as set forth in claim 1 whereinsaid compound of Formula II corresponds to Formula IIA: ##STR134##wherein: --A--A-- represents the group --CH₂ --CH₂ -- or--CH═CH--,--B--B-- represents the group --CH₂ --CH₂ -- or an alpha- orbeta-oriented group of Formula IIIA: ##STR135## R¹ represents analpha-oriented lower alkoxycarbonyl radical, X represents two hydrogenatoms or oxo, Y¹ and Y² together represent the oxygen bridge --O--, orY¹ represents hydroxy, and Y² represents hydroxy, lower alkoxy or, if Xrepresents H₂, also lower alkanoyloxy,and salts of compounds in which Xrepresents oxo and Y² represents hydroxy-, the process comprising:contacting a solution comprising a lower alkanoic acid and a salt of alower alkanoic acid with a compound corresponding to Formula IVA:##STR136## wherein --A--A--, R¹, --B--B--, X, Y¹ and Y² are as definedin Formula IIA and R² is lower alkylsulfonyloxy or acyloxy.
 3. A processas set forth in claim 1 wherein said compound of Formula IV is##STR137## and said compound of Formula II is ##STR138## wherein Msrepresents a methylsulfonyl radical.
 4. A process for the preparation ofa compound of Formula IV: ##STR139## wherein --A--A-- represents thegroup --CHR⁴ --CHR⁵ -- or --CR⁴ ═CR⁵ --R³, R⁴ and R⁵ are independentlyselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano,aryloxy, R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical, --B--B-- represents the group --CHR⁶ --CHR⁷ --or an alpha- or beta-oriented group: ##STR140## where R⁶ and R⁷ areindependently selected from the group consisting of hydrogen, halo,lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, and R⁸ and R⁹ areindependently selected from the group consisting of hydrogen, halo,lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy or R⁸ and R⁹ togethercomprise a carbocyclic or heterocyclic ring structure, or R⁸ or R⁹together with R⁶ or R⁷ comprise a carbocyclic or heterocyclic ringstructure fused to the pentacyclic D ring, and R² is loweralkylsulfonyloxy or acyloxy or a halidethe process comprising: reactinga lower alkylsulfonylating or acylating reagent or a halide generatingagent with a compound of Formula V ##STR141## wherein --A--A--, R¹, R³,--B--B--, R⁸, and R⁹ are as defined above.
 5. A process as set forth inclaim 4 wherein said compound of Formula IV corresponds to Formula IVA:##STR142## wherein: --A--A-- represents the group --CH₂ --CH₂ -- or--CH═CH--,R¹ represents an alpha-oriented lower alkoxycarbonyl radical,R² represents lower alkylsulfonyloxy or acyloxy, --B--B-- represents thegroup --CH₂ --CH₂ -- or an alpha- or beta-oriented group: ##STR143## Xrepresents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge --O--, or Y¹ represents hydroxy, and Y² representshydroxy, lower alkoxy or, if X represents H₂, also lower alkanoyloxy,andsalts of compounds in which X represents oxo and Y² represents hydroxy-,the process comprising: reacting a lower alkylsulfonyl or acyl halide inthe presence of a hydrogen halide scavenger with a compoundcorresponding to the formula: ##STR144## wherein --A--A--, R¹, --B--B--,X, Y¹, and Y² are as defined in Formula IVA.
 6. A process as set forthin claim 4 wherein said compound of Formula IV is ##STR145## and saidcompound of Formula V is ##STR146## wherein Ms represents amethylsulfonyl radical.
 7. A process for the preparation of a compoundof Formula V: ##STR147## wherein --A--A-- represents the group --CHR⁴--CHR⁵ -- or --CR⁴ ═CR⁵ --R³, R⁴ and R⁵ are independently selected fromthe group consisting of hydrogen, halo, hydroxy, lower alkyl, loweralkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy, R¹represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical, --B--B-- represents the group --CHR⁶ --CHR⁷ -- or an alpha- orbeta-oriented group: ##STR148## where R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen, halo, lower alkoxy,acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, and R⁸ and R⁹ are independently selectedfrom the group consisting of hydrogen, halo, lower alkoxy, acyl,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,acyloxyalkyl, cyano, aryloxy, or R⁸ and R⁹ together comprise acarbocyclic or heterocyclic ring structure, or R⁸ or R⁹ together with R⁶or R⁷ comprise a carbocyclic or heterocyclic ring structure fused to thepentacyclic D ring;the process comprising: reacting a compound ofFormula VI with an alkali metal alkoxide corresponding to the formulaR¹⁰ OM wherein M is alkali metal and R¹⁰ O-- corresponds to the alkoxysubstituent of R¹, said compound of Formula VI having the structure:##STR149## wherein --A--A--, R³, --B--B--, R⁸, and R⁹ are as definedabove.
 8. A process as set forth in claim 7 wherein the compound ofFormula V corresponds to the formula: ##STR150## wherein --A--A--represents the group --CH₂ --CH₂ -- or --CH═CH--,R¹ represents analpha-oriented lower alkoxycarbonyl radical, --B--B-- represents thegroup --CH₂ --CH₂ -- or an alpha- or beta-oriented group: ##STR151## Xrepresents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge --O--, or Y¹ represents hydroxy, and Y² representshydroxy, lower alkoxy or, if X represents H₂, also lower alkanoyloxy,andsalts of compounds in which X represents oxo and Y² represents hydroxy-,the process comprising: reacting a compound of Formula VIA with analkali metal alkoxide corresponding to the formula R¹⁰ OM in thepresence of an alcohol having the formula R¹⁰ OH, wherein M is alkalimetal and R¹⁰ O-- corresponds to the alkoxy substituent of R¹, saidcompound of Formula VI having the structure: ##STR152## wherein--A--A--, --B--B--, Y¹, Y² and X are as defined in Formula VA.
 9. Aprocess as set forth in claim 7 wherein the compound of Formula V is##STR153## and the compound of Formula VI is ##STR154##10.
 10. A processas set forth in claim 7 wherein cyanide ion is formed as a by-product ofthe reaction, the process further comprising removal of cyanide ion fromthe reaction zone during the reaction to reduce the extent of anyreaction of cyanide ion with the product of Formula V.
 11. A process asset forth in claim 10 wherein cyanide ion is removed from the reactionby precipitation with a precipitating agent.
 12. A process as set forthin claim 11 wherein said reaction is carried out in a solvent medium,and said precipitating agent comprises a salt comprising a cation whichforms a cyanide compound of lower solubility in said medium than thesolubility of the precipitating agent therein.
 13. A process as setforth in claim 12 wherein said cation is selected from the groupconsisting of alkaline earth metal ions and transition metal ions.
 14. Aprocess as set forth in claim 3 wherein R² is alkylsulfonyloxy, and saidprocess comprises contacting a solution comprising a lower alkanoic acidand an acid salt of a lower alkanoic acid with said compound of FormulaIV.
 15. A process as set forth in claim 3 wherein R² is mesyloxy, andsaid process comprises contacting a solution comprising formic acid andpotassium formate with said compound of Formula IV.
 16. A process as setforth in claim 3 wherein said compound of Formula IV is heated in anorganic solvent.
 17. A process as set forth in claim 4 wherein saidhalide generating agent is selected from the group consisting of thionylhalide, sulfuryl halide, and oxalyl halide.
 18. A process as set forthin claim 6 wherein said process comprises reacting said compound ofFormula V with a lower alkylsulfonyl or acyl halide in the presence of ahydrogen halide scavenger.
 19. A process as set forth in claim 6 whereinsaid compound of Formula V is reacted with methanesulfonyl chloride. 20.A process as set forth in claim 6 wherein the initial concentration ofsaid compound of Formula V is between about 5% to about 50% by weight.21. A process as set forth in claim 9 wherein the process comprisesreacting said compound of Formula VI with an alkali metal alkoxidecorresponding to the formula R¹⁰ OM in the presence of an alcohol havingthe formula R¹⁰ OH, wherein M is an alkali metal and R¹⁰ O-- correspondsto the alkoxy substituent of R¹.
 22. A process as set forth in claim 9wherein said alkali metal alkoxide is selected from potassium methoxideand sodium methoxide.
 23. A process as set forth in claim 9 wherein theinitial concentration of said compound of Formula VI is between about 2%to about 12% by weight.
 24. A process as set forth in claim 9 whereinthe reaction time is between about 4 hours to 24 hours.
 25. A process asset forth in claim 9 wherein the reaction occurs in the presence of analcohol and the ratio of the initial molar concentration of the alcoholto the initial molar concentration of the compound of Formula VI isbetween about 0.5 to about
 4. 26. A process for the preparation of acompound corresponding to Formula II: wherein:--A--A-- represents thegroup --CHR⁴ --CHR⁵ -- or --CR⁴ ═CR⁵ -- R³, R⁴ and R⁵ are independentlyselected from the group consisting of hydrogen, halo, hydroxy, loweralkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano,aryloxy, R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical, --B--B-- represents the group --CHR⁶ --CHR⁷ --or an alpha- or beta-oriented group: ##STR155## where R⁶ and R⁷ areindependently selected from the group consisting of hydrogen, halo,lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, and R⁸ and R⁹ areindependently selected from the group consisting of hydrogen, halo,lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, or R⁸ and R⁹ togethercomprise a carbocyclic or heterocyclic ring structure,the processcomprising: preparing a compound of Formula V ##STR156## wherein--A--A--, R¹, R³, --B--B--, R⁸, and R⁹ are as defined above by reactinga compound of Formula VI with an alkali metal alkoxide corresponding tothe formula R¹⁰ OM wherein M is alkali metal and R¹⁰ O-- corresponds tothe alkoxy substituent of R¹, said compound of Formula VI having thestructure: ##STR157## wherein --A--A--, R³, --B--B--, R⁸, and R⁹ are asdefined above; without isolating said compound of Formula V in purifiedform, reacting said compound of Formula V with a loweralkylsulfonylating or acylating reagent or a halide generating agent toproduce a compound of Formula IV ##STR158## wherein --A--A--, R¹, R³,--B--B--, R⁸, and R⁹ are as defined above, and R² is alkylsulfonyloxy,acyloxy leaving group or halide; without isolating said compound ofFormula IV in purified form, removing the 11α-leaving group therefrom byreaction with a reagent for abstraction thereof to produce said compoundof Formula II.